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Abstract: The ability to appropriately respond to proteotoxic stimuli is a major determinant of longevity and involves induction of various heat shock response (HSR) genes, which are essential to cope with cellular and organismal insults throughout lifespan. The activity of NAD+-dependent deacetylase Sir2, originally discovered in yeast, is known to be essential for effective HSR and longevity. Our previous work on HSR in Daphnia pulicaria indicated a drastic reduction of the HSR in older organisms. In this report we investigate the role of Sir2 in regulating HSR during the lifespan of D. pulicaria. We cloned Daphnia Sir2 open reading frame (ORF) to characterize the enzyme activity and confirmed that the overall function of Sir2 was conserved in Daphnia. The Sir2 mRNA levels increased while the enzyme activity declined with age and considering that Sir2 activity regulates HSR, this explains the previously observed age-dependent decline in HSR. Finally, we tested the effect of Sir2 knockdown throughout adult life by using our new RNA interference (RNAi) method by feeding. Sir2 knockdown severely reduced both the median lifespan as well as significantly increased mortality following heat shock. Our study provides the first characterization and functional study of Daphnia Sir2

Telomerase activity in Daphnia.

<p>A) Telomeric repeat amplification protocol (TRAP) assay of <i>D</i>. <i>pulex</i> extracts. The amount of cell extract used is indicated above each lane. HI: Heat Inactivated, CE: Cell Extract. TSR8 CT: positive control for PCR step. B) Quantification of the TRAP Assay in 1A. Student T-test was performed, p values are as follows: * = 6.15x10^-6, ** = 6.63x10^-6 (n = 3).</p

Telomerase from <i>Daphnia</i> embryos shows high processivity.

<p>A) TRAP assay from egg stage 1 embryos and 1 week old adults of <i>D</i>. <i>pulex</i> and <i>D</i>. <i>pulicaria</i>. 250 ng of <i>Daphnia</i> extract was used in each lane. B) Quantification of the TRAP assay displayed in 2 A. C) Quantification of the processivity of telomerase from each displayed sample. The p values calculated from student T-tests are as follows: * = 6.15x10^-6, ** = 5.56x10^-6, *** = 0.0005,**** = 1.5x10^-5; # = 0.0002, ## = 0.0002 (n = 4).</p

Comparison of telomerase activity at different ages in <i>D</i>. <i>pulex</i> and <i>D</i>. <i>pulicaria</i>.

<p>A) TRAP assay was performed using <i>Daphnia</i> extracts prepared at indicated ages. Lanes 1–3: <i>D</i>. <i>pulex</i>, lanes 4–6: <i>D</i>. <i>pulicaria</i>. B) Quantification of the TRAP assay displayed in 4 A. C) Quantification of the processivity of telomerase from each displayed sample. Student T-tests were performed, and p values are as follows * = 0.0005, ** = 0.0006, *** = 0.002, # = 0.001, ## = 0.034, ### = 0.0132, #### = 0.029 (n = 4).</p

Telomeric repeat sequence from <i>Daphnia</i> and sequence alignment of <i>Daphnia</i> telomerase (dTERT) with telomerase proteins from other species.

<p>A) Telomeric repeat sequence of <i>Daphnia</i> and other species. <i>H</i>. <i>americanus</i>: <i>Homarus americanus</i>—Lobster, <i>G</i>. <i>pulex</i>: <i>Gammarus pulex</i>—Freshwater amphipod, <i>B</i>. <i>mori</i>: <i>Bombyx mori—</i>Silkworm/moth, <i>A</i>. <i>viridus</i>: <i>Amaranthus viridus</i>—Beetle, <i>M</i>. <i>musculus</i>: <i>Mus musculus</i>—Mouse, <i>H</i>. <i>sapiens</i>: <i>Homo sapiens</i>—Human. B) Identity and similarity percentages of dTERT with TERT from other species. C) <i>D</i>. <i>pulex</i> telomerase reverse transcriptase (DTERT) sequence alignment for the RNA-binding and reverse transcriptase domains. Green Boxes: conserved residues within the RNA binding domain. Black Boxes: conserved residues within the reverse transcriptase domain. Blue boxes: residues within the reverse transcriptase domain that are essential for nucleic acid binding.</p

Life spans and telomere lengths in <i>D</i>. <i>pulex</i> (RW20) and <i>D</i>. <i>pulicaria</i> (LakeXVI-clone11).

<p>A) Survivorship curves of clones RW20 (open squares) and XVI-11 (black circles). Error bars show age-specific standard errors from Kaplan-Meier survival probability estimates. B) Terminal Restriction Fragment (TRF) assay was performed to estimate the average telomere length of the <i>Daphnia</i> at various ages. The age in weeks is indicated above each lane. Lanes 1–3: <i>D</i>. <i>pulex</i>, lanes 4–6: <i>D</i>. <i>pulicaria</i>. M1 and M2: molecular weight markers; lambda HindIII digest and lambda BstEII digest respectively. C) Quantification of 5 A, error bars indicate standard deviation. Student T-tests were performed and p values are as follows: * = 1.03x10^-5, ** = 0.007, *** = 2.48x10^-5 (n = 3).</p