Effect of anoxia on nucleotide metabolism in encysted embryos of the brine shrimp

Underanoxia, Artemia franciscana embryos downregulate metabolic processes and approach an ametabolic state. Entrance into this quiescent state is accompanied by a profound acidification of the intracellular space, and more than two decades of research now clearly demonstrates that this acidification is critical to metabolic downregulation in anoxic embryos. However, the proximal mechanisms responsible for the pH shift remain largely unidentified. Here, we report evidence demonstrating expression of the V-ATPase in encysted embryos and present an argument for its involvement in the intracellular acidification induced by anoxia. We identified a single B-subunit cDNA sharing the greatest degree of sequence similarity with 'generalisttype' homologues from mammals (brain-type) and invertebrates. Quantitative analysis of B-subunit mRNA demonstrates differential expression throughout early development, and western blot analyses confirm the expression of at least six V-ATPase subunits in both heavy membranes and microsomal vesicles. The critical need for proton pumping during the anoxia-tolerant stage of development is demonstrated by incubation with the VATPase inhibitor bafilomycin A 1 , which halts embryonic development. Importantly, net proton flux from VATPase-acidified compartments to the surrounding cytoplasm is likely under anoxia and may significantly contribute to the enigmatic acidification critical to quiescence.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

V-ATPase expression during development ofArtemia franciscanaembryos: potential role for proton gradients in anoxia signaling

Covi

Hand

2005

“…Their tolerance to anoxia in this quiescent state (Fig. 1) is impressive but will not be reviewed here (see Stocco et al, 1972;Busa and Crowe, 1983;Clegg and Jackson, 1989;Hand, 1991;Hand and Hardewig, 1996;Warner and Clegg, 2001;Clegg, 2011;Hand et al, 2011). Annual killifish inhabit ephemeral ponds in regions of Central and South America and Africa that experience distinct dry and rainy seasons (Polacik and Podrabsky, 2015).…”

Section: Diapause During the Life Cycle Of Artemia Franciscanamentioning

confidence: 99%

Physiological strategies during animal diapause: lessons from brine shrimp and annual killifish

Podrabsky

Hand

2015

Diapause is a programmed state of developmental arrest that typically occurs as part of the natural developmental progression of organisms that inhabit seasonal environments. The brine shrimp Artemia franciscana and annual killifish Austrofundulus limnaeus share strikingly similar life histories that include embryonic diapause as a means to synchronize the growth and reproduction phases of their life history to favorable environmental conditions. In both species, respiration rate is severely depressed during diapause and thus alterations in mitochondrial physiology are a key component of the suite of characters associated with cessation of development. Here, we use these two species to illustrate the basic principles of metabolic depression at the physiological and biochemical levels. It is clear that these two species use divergent molecular mechanisms to achieve the same physiological and ecological outcomes. This pattern of convergent physiological strategies supports the importance of biochemical and physiological adaptations to cope with extreme environmental stress and suggests that inferring mechanism from transcriptomics or proteomics or metabolomics alone, without rigorous follow-up at the biochemical and physiological levels, could lead to erroneous conclusions.

“…In this dehydrated state, damage to proteins, membranes and other macromolecular structures is prevented by the presence of a high level of trehalose and a number of stress proteins (MacRae, 2003). This stress resistance is shared by a number of other anhydrobiotic animals, including rotifers and tardigrades; however, Artemia embryos have the almost unique ability to survive for extended periods in the fully hydrated state in the absence of molecular oxygen (Clegg, 1992;Dutrieu and Chrestia-Blanchine, 1966;Stocco et al, 1972). Even after four years of continuous anoxia at 20-23°C, a hatch rate of at least 60% can be achieved when oxygen is restored.…”

Section: Introductionmentioning

confidence: 99%

Ametabolic embryos of Artemia franciscana accumulate DNA damage during prolonged anoxia

McLennan

2009

SUMMARYEncysted embryos of the brine shrimp Artemia franciscana are able to survive prolonged periods of anoxia even when fully hydrated. During this time there is no metabolism, raising the question of how embryos tolerate spontaneous, hydrolytic DNA damage such as depurination. When incubated at 28°C and 40°C for several weeks, hydrated anoxic embryos were found to accumulate abasic sites in their DNA with k=5.8ϫ10 -11 s -1 and 2.8ϫ10 -10 s -1 , respectively. In both cases this is about 3-fold slower than expected from published observations on purified DNA. However, purified calf thymus DNA incubated under similar anoxic conditions at pH 6.3, the intracellular pH of anoxic cysts, also depurinated more slowly than predicted (about 1.7-fold), suggesting that cysts may in fact accumulate abasic sites only slightly more slowly than purified DNA. Upon reoxygenation of cysts stored under N 2 for 30 weeks at 28°C, the number of abasic sites per 10 4 bp DNA fell from 21.1±4.0 to 9.8±2.0 by 12 h and to 6.2±2.1 by 24 h. Larvae hatched after 48 h and 72 h had only 0.59±0.17 and 0.48±0.07 abasic sites per 10 4 bp, respectively, suggesting that repair of these lesions had largely taken place before hatching commenced. Thus, unlike bacterial spores, Artemia cysts appear to have no specific protective mechanism beyond what might be afforded by chromatin structure to limit spontaneous depurination, and rely on the repair of accumulated lesions during the period between reoxygenation and hatching.