The Effects of MsrA and MsrB in Anoxia Tolerance in Aging Drosophila melanogaster Nirthieca Suthakaran Advisor: Dr. David Binninger Dr. Evelyn Frazier

Oxygen deprivation What is Anoxia?

Dangers of Anoxia Reoxygenation Effect Introducing oxygen back into the system Burst of reactive oxygen species (ROS) Superoxide anion (O 2 - ) Cellular Damage Hydrogen peroxide (H 2 O 2 ) Methionine Oxidation

DROSOPHILA

Methionine Sulfoxide Reductase Oxidation Reduction

Does MsrA and MsrB play a role in responding to anoxic conditions? Research Question

Msr Single Deletion Fly Lines GenotypeMsrAMsrB WT60Present A90Not PresentPresent B54PresentNot Present

Research Strategy 2) Flies were placed in the DAM holder for 10 minutes of stabilization 4) Flies were placed into the DAM system for 5 hours 3) Flies were placed into the anoxia tank for 1 hour. 1) Individual flies for each genotype were placed into each of the 32 tubes in the Drosophila Activity Monitor (DAM) holder. 5) Analyzed and averaged the recovery times recorded by the DAM system

Preliminary Data: Longer Recovery Time in Older Aged Flies WT60: Wild-type control (MsrA + MsrB + ) A90: MsrA Loss of Function (MsrA - MsrB + ) B54: MsrB Loss of Function (MsrA + MsrB - ) AB46: Msr Double Loss of Function (MsrA - MsrB - ) Legend * * * * * * *

Aging : days (young age) days (middle age) days (old age) 32 flies 10 min Normoxia 32 flies 1 hour Anoxia 32 flies 1 hour anoxia + 5 hours Reoxygenation Will Msr single LOF mutant flies show increased susceptibility to anoxia as they approach senescence (60-65 days)? Research Question #1

Single Deletion Line Results The WT60 line recover significantly later as they approach senescence, while the MsrA and MsrB mutants reach maximum recovery time at middle age (40-45 days old). C.C. A.C.B. A.B.C.

Does a knock down of the single LOF Msr genes via RNAi show increased susceptibility to anoxia as they approach senescence (60-65 days)? Research Question #2 RNAi lines: UAS-Gal4 system Main Driver Used: Actin Driver

Creating RNAi Lines

Results for RNAi Lines The RNAi-A and RNAi-B knockdown lines reach maximum average recovery time after anoxic stress at middle age (40-45 days old). A. B. C. * * * * *

The MsrA + MsrB + (WT60) line recovers significantly later as the flies approached senescence (60-65 days old). The MsrA and MsrB single deletion mutants reach maximum recovery time at middle age (40-45 days old). Unexpectedly, the day old MsrA and MsrB single deletion lines recover faster than the day old single deletion lines and are indistinguishable from the MsrA + MsrB + (WT60) line. Summary & Significance

Similar results are not seen in their respective RNAi lines, although there are a few discrepancies because the RNAi-lines may contain residual forms of the MsrA or MsrB gene. As the flies approached senescence (60-65 days), the decrease in average recovery time among the MsrA and MsrB deletion lines, indicate that there may be a novel compensatory mechanism causing this. Summary & Significance

ACKNOWLEDGEMENTS Dr. David Binninger Dr. Evelyn Frazier Dr. John Nambu Ramon Garcia and Chelsea Bennice Dr. Sarah Milton Lindsay Bruce Lab mates Honors Program and Peers National Institute of Health Grant FAU Undergraduate Research Grant

Questions? Questions?