Alternative mitochondrial respiratory chains from two crustaceans: Artemia franciscana nauplii and the white shrimp, Litopenaeus vannamei

Abstract

Mitochondrial ATP is synthesized by coupling between the electron transport chain and complex V. In contrast, physiological uncoupling of these processes allows mitochondria to consume oxygen at high rates without ATP synthesis. Such uncoupling mechanisms prevent reactive oxygen species overproduction. One of these mechanisms are the alternative redox enzymes from the mitochondrial respiratory chain, which may help cells to maintain homeostasis under stress independently of ATP synthesis. To date, no reports have been published on alternative redox enzymes in crustaceans mitochondria. Specific inhibitors were used to identify alternative redox enzymes in mitochondria isolated from Artemia franciscana nauplii, and the white shrimp, Litopenaeus vannamei. We report the presence of two alternative redox enzymes in the respiratory chain of A. franciscana nauplii, whose isolated mitochondria used glycerol-3-phosphate as a substrate, suggesting the existence of a glycerol-3-phosphate dehydrogenase. In addition, cyanide and octyl-gallate were necessary to fully inhibit this species' mitochondrial oxygen consumption, suggesting an alternative oxidase is present. The in-gel activity analysis confirmed that additional mitochondrial redox proteins exist in A. franciscana. A mitochondrial glycerol-3-phosphate dehydrogenase oxidase was identified by protein sequencing as part of a branched respiratory chain, and an alternative oxidase was also identified in this species by western blot. These results indicate different adaptive mechanisms from artemia to face environmental challenges related to the changing levels of oxygen concentration in seawater through their life cycles. No alternative redox enzymes were found in shrimp mitochondria, further efforts will determine the existence of an uncoupling mechanism such as uncoupling proteins.

Identification of the alternative oxidase gene and its expression in the copepod Tigriopus californicus

“…We have shown that AOX is in the genomes of multiple species of copepods from around the world that inhabit a wide variety of ecological niches (Table 2.) The first reports of AOX from arthropods were in the brine shrimp Artemia franciscana and putative sequences from other members of the Chelicerata, Hexapoda, and Crustacea (Rodriguez-Armenta et al, 2018;McDonald and Gospodaryov, 2018). It was previously hypothesized that AOX was not present in arthropods due to a gene loss event (McDonald et al, 2009), however, the above data refute this hypothesis.…”

“…Heterologously expressed AOX proteins from Ciona intestinalis and Crassostrea gigas are 35-42 kDa in size (Hakkaart et al, 2006;Robertson et al, 2016). The AOX protein of Artemia franciscana is between 25 and 37 kDa on Western blots using the anti-AOX Sauromatum guttatum monoclonal antibody (Rodriguez-Armenta et al, 2018). It is expected that the food source (Spirulina) would contain AOX as it is an algae, however, the AOX antibody did not recognize a protein in this sample (Figure A.3).…”

“…The bulk of information about the AOX of animals exists at the level of genes or transcripts. The only reports of naturally occurring AOX proteins present in animals are from the brine shrimp Artemia franciscana and the Pacific oyster Crassostrea gigas; AOX proteins in these species are ~35 kDa in size (Rodriguez-Armenta et al, 2018).…”

The Tardigrade Hypsibius Exemplaris as an Emerging Model to Study the Mitochondrial Alternative Oxidase at Animal Organismal Level

“…In the case of C. gigas, AOX contribution to the oyster adjustments to short-term hypoxia and re-oxygenation was considered in isolated mitochondria [17] but the presented data do not allow for clear conclusions as the observed AA-resistant respiration was not shown to be eliminated by AOX inhibitor, for example by benzohydroxamic acid (BHAM) [35]. Moreover, to observe the enzyme activity in the shrimp isolated mitochondria the authors applied an uncoupler to increase the recorded oxygen uptake rate [32].…”

“…The second one was heterologously expressed in mitochondria of cultured human cells [9,[20][21] as well as in the fruit y Drosophila (Sophophora) melanogaster [22] [23][24][25][26][27] and mouse [9,12,[28][29]. Additionally, some experimental data concerning AOX protein are available for crustaceans, the brine shrimp Artemia franciscana [30], the white shrimp Litopenaeus vannamei [31] [32] and the marine copepod Tigriopus californicus [33] [34]. In the case of both the shrimps the data concerns AOX activity in isolated mitochondria whereas in the case of the copepod amounts of AOX mRNA and protein in various life stages of the animal and under stress temperature conditions were studied.…”