Abstract
Caloric restriction (CR) produces clear phenotypic effects within and between generations of the model crustacean Daphnia magna. We have previously established that micro-RNAs and cytosine methylation change in response to CR in this organism, and we demonstrate here that CR has a dramatic effect on gene expression. Over 6,000 genes were differentially expressed between CR and well-fed D. magna, with a bias towards up-regulation of genes under caloric restriction. We identified a highly expressed haemoglobin gene that responds to CR by changing isoform proportions. Specifically, a transcript containing three haem-binding erythrocruorin domains was strongly down-regulated under CR in favour of transcripts containing fewer or no such domains. This change in the haemoglobin mix is similar to the response to hypoxia in Daphnia, which is mediated through the transcription factor hypoxia-inducible factor 1, and ultimately the mTOR signalling pathway. This is the first report of a role for haemoglobin in the response to CR. We also observed high absolute expression of superoxide dismutase (SOD) in normally fed individuals, which contrasts with observations of high SOD levels under CR in other taxa. However, key differentially expressed genes, like SOD, were not targeted by differentially expressed micro-RNAs. Whether the link between haemoglobin and CR occurs in other organisms, or is related to the aquatic lifestyle, remains to be tested. It suggests that one response to CR may be to simply transport less oxygen and lower respiration.
Highlights
Caloric restriction (CR) is the reduction in dietary intake of calories without undernutrition (Koubova & Guarente, 2003)
In part 1, we focus on the haemoglobin response that emerged from the DTU analysis with support from gene expression results; part 2, discusses the endoplasmic-reticulum stress response results from Gene set enrichment analysis (GSEA); part 3, down-regulation of super-oxide dismutase is a gene-level response identified in DESeq2; part 4, shows overlap in GO and GSEA down-regulated in CR processes; part 5, asks why differential methylation identified in response to CR in the same clone (Hearn, Pearson, et al, 2019) did not impact upon gene expression; part 6 examines miRNA correlations that were biased towards genes up-regulated in CR
By contrasting differential transcript usage between CR and normal food (NF) we showed that the haemoglobin isoform mix of a highly-expressed D. magna Hb gene is reduced for isoforms containing erythrocruorin domains
Summary
Caloric restriction (CR) is the reduction in dietary intake of calories without undernutrition (Koubova & Guarente, 2003). Most notably an increase in longevity has been observed in various arthropods, rodents, yeast, and possibly in humans (Heilbronn & Ravussin, 2003; Kapahi, Kaeberlein, & Hansen, 2017; Lakowski & Hekimi, 1998; Redman & Ravussin, 2011; Sohal & Weindruch, 1996; Walford, Harris, & Weindruch, 1987). This occurs through CRmediated delays in the onset of processes and diseases associated with ageing (Koubova & Guarente, 2003; Most, Tosti, Redman, & Fontana, 2017). Despite the popularity of Daphnia magna for studying life-history traits and the effects of environmental stressors (Boersma, Spaak, & De Meester, 1998; Garbutt & Little, 2014, 2017; Lampert, 1987; Latta IV, Frederick, & Pfrender, 2011; Mitchell & Lampert, 2000; Orsini et al, 2016), the gene-level response to CR has not previously been studied in this species
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