Metabolic depression is delayed and mitochondrial impairment averted during prolonged anoxia in the ghost shrimp, Lepidophthalmus louisianensis (Schmitt, 1935)

Abstract

Lepidophthalmus louisianensis burrows deeply into oxygen-limited estuarine sediments and is subjected to extended anoxia at low tides. Large specimens (> 2 g) have a lethal time for 50% mortality (LT₅₀) of 64 h under anoxia at 25 °C. Small specimens (< 1 g) have a significantly higher LT₅₀ of 113 h, which is the longest ever reported for a crustacean. Whole body lactate levels rise dramatically under anoxia and exceed 120 μmol g.f.w.^− 1 by 72 h. ATP, ADP, and AMP do not change during 48 h of anoxia, but arginine phosphate declines by over 50%. Thus arginine phosphate may help stabilize the ATP pool. Surprisingly, when compared to the aerobic resting rate, ATP production under anoxia declines only moderately during the first 12 h, and drops to only about 30% between 12 and 48 h. Finally, after 48 h of anoxia, a major metabolic depression to less than 5% occurs. Downregulation of metabolism is delayed in L. louisianensis compared to many invertebrates that exhibit facultative anaerobiosis. Bioenergetic constraints as a result of eventual metabolic depression lead to ionic disturbances like calcium overload and compromised membrane potential of mitochondria. Because these phenomena trigger apoptosis in mammalian species, we evaluated the susceptibility of ghost shrimp mitochondria to opening of the mitochondrial permeability transition pore (MPTP) and associated damage. Energized mitochondria isolated from hepatopancreas possess a pronounced capacity for calcium uptake. Exogenous calcium does not stimulate opening of the MPTP, which potentially could reduce cell death during prolonged anoxia.

Introduction

The ghost shrimp, Lepidophthalmus louisianensis (formerly Callianassa jamaicense) is a burrowing decapod crustacean of the infraorder Thalassinidea (Manning and Felder, 1991) that is commonly found in estuaries along the northern Gulf of Mexico. Dense populations of L. louisianensis are found in low-energy beaches, back-beach ponds, estuarine tidal flats, and tidal streams where they construct permanent and semi-permanent galleries up to several meters deep into the anoxic, sulfide-rich sediment (Willis, 1942, Felder, 1978, Britton and Morton, 1989, Felder and Griffis, 1994, Felder, 2001). When the tide recedes, the burrow water can become severely hypoxic or anoxic within hours, and during peak low tides or storm surges, these hypoxic conditions can last for days (Felder, 1979). The main objective of the present study is to evaluate a suite of physiological mechanisms that underlie the prolonged anoxia tolerance of this species.

Low resting metabolic rates and critical PO₂ values (ambient PO₂ at which oxygen consumption decreases linearly with ambient oxygen, P_crit) have been observed in L. louisianensis by Felder (1979) and Neotrypaea californiensis (formerly Callianassa californiensis) by Thompson and Pritchard (1969) and Torres et al. (1977) as well as other thalassinideans (see Stanzel and Finelli, 2004, Atkinson and Taylor, 2005). As ambient PO₂ approaches the respective P_crit value, crustaceans recruit fermentative pathways to provide additional ATP. Anaerobic glycolysis leading to the accumulation of lactate is probably the only source of ATP provision during anoxia in Crustacea (Albert and Ellington, 1985, Grieshaber et al., 1994). Generally, lactate fermentation is associated with low tolerance (short-term survival) under anoxia because the substrate, mostly glycogen, is rapidly consumed (Hochachka, 1980, Hagerman, 1998). This limitation may explain the inability of most decapods to withstand severe hypoxia or anoxia for more than a few hours. Unlike its decapod relatives, L. louisianensis and other thalassinid shrimps survive anoxic conditions in the laboratory for several days (Thompson and Pritchard, 1969, Felder, 1979, Zebe, 1982). Though very small amounts of alanine, aspartate, glutamate, succinate, and malate have been reported in N. californiensis, l-lactate is the principal metabolic end product measured in that species and in Upogebia pugettensis (Pritchard and Eddy, 1979, Zebe, 1982). Unpublished observations suggest that lactate accumulates in L. louisianensis under hypoxia (Felder et al., 1995, Bourgeois and Felder, 2001).

Given that L. louisianensis survives anoxia nearly twice as long as C. californiensis and U. pugettensis (Felder, 1979), even at warmer temperatures, it is possible that anaerobic end products in addition to lactate are accumulated. Freshwater snails, mussels, oysters, and lugworms have anaerobically functioning mitochondria that accumulate succinate and the volatile fatty acids (VFAs) propionate and acetate via the malate dismutase pathway (see Grieshaber et al., 1994, van Hellemond et al., 1995, Tielens et al., 2002). Unlike aerobic mitochondria, which utilize ubiquinone for electron transfer to Complex III, the transfer of electrons to fumarate under anoxia is accomplished with rhodoquinone (van Hellemond et al., 1995, Tielens et al., 2002). Rhodoquinone is essential for anaerobic accumulation of succinate, propionate, acetate, and other VFAs including isovalerate, methylbutyrate, and isobutyrate (Lahoud et al., 1971, Hochachka, 1980, Holst and Zebe, 1986, Kita, 1992, Tielens, 1994). Thus we measured the amount of rhodoquinone present in the mitochondria of L. louisianensis to gain insight into the capacity for production of non-lactate end products.

Based on the strong positive correlation between the anoxia tolerance of species and their capacity for acute metabolic depression (Hand, 1998, Hochachka and Lutz, 2001), we predicted that under anoxia L. louisianensis would reduce metabolic rate severely and quickly (below 10% of the aerobic value), which would lower the rate of ATP consumption and conserve carbohydrate fuels. To estimate the degree of metabolic depression, we chose to estimate the rate of ATP turnover under anoxia from end product accumulation and arginine phosphate use, and then express the value as a percentage of the resting aerobic rate as calculated from oxygen consumption.

Such bioenergetic constraints under anoxia inevitably lead to ionic disturbances like calcium overload in cells, and the compromise of mitochondrial membrane potential (ΔΨ) will occur with extended time (Hochachka, 1986, Hand and Menze, 2008). When ATP availability drops so low that active ion transport across membranes cannot keep up with passive ion leak, then dissipation of ion gradients can take place (Covi and Hand, 2005, Covi et al., 2005, Covi and Hand, 2007). These phenomena signal initiation of apoptosis in mammals (Kroemer et al., 2007, Hand and Menze, 2008). Because of the remarkable tolerance to anoxia by L. louisianasis, we evaluated the susceptibility of mitochondria of this ghost shrimp to opening of the mitochondrial permeability transition pore (MPTP). If mammalian mitochondria are exposed to elevated calcium in the presence of phosphate, especially when accompanied by depletion of adenine nucleotides and reduced ΔΨ across the inner membrane (Petronilli et al., 1993a), a large swelling occurs that is associated with uncoupled respiration and cytochrome-c release (Haworth and Hunter, 1979, Hunter and Haworth, 1979, Gunter and Pfeiffer, 1990, Halestrap et al., 2000, Kroemer et al., 2007). These phenomena are due to an acute increase in permeability of the inner mitochondrial membrane known as the permeability transition (Hunter et al., 1976, Bernardi, 1996, Bernardi et al., 2006;). Matrix swelling then causes the rupture of the outer mitochondrial membrane and release of numerous pro-apoptotic factors from the intermembrane space (Green and Reed, 1998, Zamzami and Kroemer, 2001, Green and Kroemer, 2004, Saelens et al., 2004, Bernardi et al., 2006). Surprisingly, Menze et al. 2005b noted that the MPTP does not open in response to high calcium in mitochondria from another anoxia-tolerant crustacean, Artemia franciscana, so we hypothesized that the pore may be refractory to anoxia-induced activators in ghost shrimp. As proposed recently (Hand and Menze, 2008), a character trait like prolonged tolerance to anoxia may be a consequence, to some degree, of specific features of apoptosis operative across species. For example, a modest elevation in calcium may trigger apoptosis in mammals, whereas severe energy limitation may not initiate cell death in certain non-mammalian species. Functional trade-offs in the predisposition to environmental tolerance may have occurred in parallel with the evolution of diversified pathways for cell death in eukaryotic organisms.