Project Title

The Effect of Chronic Mild Intermittent Hypoxia, and 2, 4-Dinitrophenol on Longevity and Gene Expression in Daphnia magna

Authors' Affiliations

Millicent Nkiruka Ekwudo, Department of Biological Sciences, College of Arts and Sciences, East Tennessee State University, Johnson City, TN Lev Yampolsky, Department of Biological Sciences, College of Arts and Sciences, East Tennessee State University, Johnson City, TN

Faculty Sponsor’s Department

Biological Sciences

Additional Sponsors

Patrick Bradshaw, Aruna Kilaru

Type

Oral Competitive

Classification of First Author

Graduate Student-Master’s

Project's Category

Ecology

Abstract Text

The mitochondria are organelles where energy in the form of ATP (Adenosine Triphosphate) is produced. During low oxygen supply (hypoxia) and mitochondrial uncoupling, ATP synthesis is reduced and AMP (Adenosine Monophosphate) accumulates in the cells. This increase in AMP: ATP ratio stimulates the AMP-Activated Kinase (AMPK) pathway, known to improve healthspan and lifespan by increasing mitochondrial biogenesis (making new mitochondria), decreasing oxidative stress and inflammation, and proteotoxicity (by degrading non-functional organelles and proteins). Here, the life/healthspan extending potential of chronic mild intermittent hypoxia (CMIH) and mitochondrial uncoupling using 2,4 -Dinitrophenol (DNP) was investigated in an emerging model organism, an aquatic crustacean, Daphnia magna. First, the effect of CMIH (4mgO2/L) on longevity in four different genotypes of Daphnia magna was investigated. All individuals were kept in similar conditions with controls in normoxia (8mgO2/L). Hypoxia was created by bubbling compressed nitrogen gas through the water twice daily. Survival was assessing through censuses conducted every 3 days and gene expression changes in response to CMIH were assessed by RNA sequencing using Oxford Nanopore Technology. Briefly, RNA was isolated from genotypes after hypoxic treatments and reverse transcribed to cDNA, libraries were multiplexed and sequenced using Oxford Nanopore MinION for 24-48 hours. Lastly, the effect of prolonged exposure to DNP on longevity was evaluated. Daphnia were chronically exposed to either 0 (control), or 0.1, 1, and 5μM of DNP. Genotypes displayed different tolerance to hypoxia and DNP treatments. Contrary to the expectations, CMIH and DNP reduced longevity, but only in genotypes from permanent ponds, while having no effect on the survival of genotypes from intermittent ponds, arguably better adapted to naturally occurring hypoxic conditions. We uncovered 11 candidate genes that were differentially expressed in these genotypes. In particular, genes involved in mTOR, p53, and sirtuin pathways showed patterns of expression consistent with protection against hypoxia. These pathways are known to regulate autophagy, apoptosis, inflammation, and cell cycle. Because our findings elucidate genotype-specific physiological and transcriptomic responses to respiratory perturbations (CMIH and DNP) we may be able to make a step towards the understanding of a model organism’s response to respiratory stress.

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The Effect of Chronic Mild Intermittent Hypoxia, and 2, 4-Dinitrophenol on Longevity and Gene Expression in Daphnia magna

The mitochondria are organelles where energy in the form of ATP (Adenosine Triphosphate) is produced. During low oxygen supply (hypoxia) and mitochondrial uncoupling, ATP synthesis is reduced and AMP (Adenosine Monophosphate) accumulates in the cells. This increase in AMP: ATP ratio stimulates the AMP-Activated Kinase (AMPK) pathway, known to improve healthspan and lifespan by increasing mitochondrial biogenesis (making new mitochondria), decreasing oxidative stress and inflammation, and proteotoxicity (by degrading non-functional organelles and proteins). Here, the life/healthspan extending potential of chronic mild intermittent hypoxia (CMIH) and mitochondrial uncoupling using 2,4 -Dinitrophenol (DNP) was investigated in an emerging model organism, an aquatic crustacean, Daphnia magna. First, the effect of CMIH (4mgO2/L) on longevity in four different genotypes of Daphnia magna was investigated. All individuals were kept in similar conditions with controls in normoxia (8mgO2/L). Hypoxia was created by bubbling compressed nitrogen gas through the water twice daily. Survival was assessing through censuses conducted every 3 days and gene expression changes in response to CMIH were assessed by RNA sequencing using Oxford Nanopore Technology. Briefly, RNA was isolated from genotypes after hypoxic treatments and reverse transcribed to cDNA, libraries were multiplexed and sequenced using Oxford Nanopore MinION for 24-48 hours. Lastly, the effect of prolonged exposure to DNP on longevity was evaluated. Daphnia were chronically exposed to either 0 (control), or 0.1, 1, and 5μM of DNP. Genotypes displayed different tolerance to hypoxia and DNP treatments. Contrary to the expectations, CMIH and DNP reduced longevity, but only in genotypes from permanent ponds, while having no effect on the survival of genotypes from intermittent ponds, arguably better adapted to naturally occurring hypoxic conditions. We uncovered 11 candidate genes that were differentially expressed in these genotypes. In particular, genes involved in mTOR, p53, and sirtuin pathways showed patterns of expression consistent with protection against hypoxia. These pathways are known to regulate autophagy, apoptosis, inflammation, and cell cycle. Because our findings elucidate genotype-specific physiological and transcriptomic responses to respiratory perturbations (CMIH and DNP) we may be able to make a step towards the understanding of a model organism’s response to respiratory stress.

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