Reversible intracellular acidification and depletion of NTPs provide a potential physiological origin for centuries of dormancy in an Antarctic freshwater copepod.

A great diversity of crustacean zooplankton found in inland and coastal waters produce embryos that settle into bottom sediments to form an egg bank. Embryos from these banks can remain dormant for centuries, creating a reservoir of genetic diversity. A large body of literature describes the ecological and evolutionary importance of zooplankton egg banks. However, literature on the physiological traits behind dormancy in crustacean zooplankton are limited. Most data on the physiology of dormancy comes from research on one species of anostracan, the brine shrimp, Artemia franciscana. Anoxia-induced dormancy in this species is facilitated by a profound and reversible acidification of the intracellular space. This acidification is accompanied by a reversible depletion of adenosine triphosphate (ATP). The present study demonstrates that acidification of the intracellular space also occurs in concert with a depletion of nucleoside triphosphates (NTPs) in the Antarctic copepod, Boeckella poppei. Like A. franciscana, the depletion of NTPs and acidification are rapidly reversed during aerobic recovery in B. poppei. These data provide the first comparative evidence that extreme dormancy under anoxia in crustacean zooplankton is associated with intracellular acidification and an ability to recover from the depletion of ATP.

Sediment from lake with missing egg bank is toxic to hatchlings of model zooplankton: A reason to consider obligate dormancy in toxicological assessment.

Over the last 60 years, valuable progress was made in the standardization of environmental monitoring with model zooplankton. However, obligate dormancy in zooplankton life cycles is not yet considered in standardized toxicology methods. Most zooplankton from coastal and inland waters use dormancy as a critical ecological strategy, and exposure to toxicants during dormancy or resurrection from dormancy alters developmental patterning and hatching success. The present study accounts for this by using both standardized and novel toxicology assays to assess the impacts of coal ash contaminated sediments and water on development, hatching, and survivorship of model zooplankton. The results demonstrate that standardized assays with rotifer and cladoceran models detect no toxicity in surface water and sediment pore water from Lake Sutton, North Carolina, USA. By contrast, novel toxicity assays with cladoceran and anostracan models demonstrate that development and larval survivorship are negatively impacted by Lake Sutton water and sediment. Embryos of Artemia franciscana display developmental patterning and hatching aberrations that match those observed in previous studies with metals when hatched in filtered surface water or pore water after a period of anoxia-induced dormancy. Larval survivorship in Daphnia magna and A. franciscana also decreases when post-diapause embryos are hatched in the presence of sediment. The effects of whole sediment on larval survivorship are not explained by coal ash impacts on water pH. These data provide an explanation for the missing egg bank and historic community restructure in Lake Sutton. The data also demonstrate a need for standardized assays that include dormant life stages.

The ultrastructure of resurrection: Post-diapause development in an Antarctic freshwater copepod.

The copepod, Boeckella poppei, is broadly distributed in Antarctic and subantarctic maritime lakes threatened by climate change and anthropogenic chemicals. Unfortunately, comparatively little is known about freshwater zooplankton in lakes influenced by the Southern Ocean. In order to predict the impact of climate change and chemicals on freshwater species like B. poppei, it is necessary to understand the nature of their most resilient life stages. Embryos of B. poppei survive up to two centuries in a resilient dormant state, but no published studies evaluate the encapsulating wall that protects theses embryos or their development after dormancy. This study fills that knowledge gap by using microscopy to examine development and the encapsulating wall in B. poppei embryos from Antarctica. The encapsulating wall of B. poppei is comprised of three layers that appear to be conserved among crustacean zooplankton, but emergence and hatching are uniquely delayed until the nauplius is fully formed in this species. Diapause embryos in Antarctic sediments appear to be in a partially syncytial mid-gastrula stage. The number of nuclei quadruples between the end of diapause and hatching. Approximately 75% of yolk platelets are completely consumed during the same time period. However, some yolk platelets are left completely intact at the time of hatching. Preservation of complete yolk platelets suggests an all-or-none biochemical process for activating yolk consumption that is inactivated during dormancy to preserve yolk for post-dormancy development. The implications of these and additional ultrastructural features are discussed in the context of anthropogenic influence and the natural environment.

Lack of dormancy to protect diversity: Decrease in diversity of active zooplankton community observed in lake with depauperate egg bank.

The study of zooplankton communities in freshwater resources under anthropogenic pressures rarely includes the simultaneous assessment of dormant embryos in bottom sediments and active life-stages in the water column. A coastal lake with a history of coal-ash contamination and disruption by hurricanes provided an ideal opportunity to demonstrate the power of examining both dormant and active zooplankton. The primary objective of this study was to evaluate changes in structure of a multicellular zooplankton community that is under simultaneous pressure from anthropogenic pollution and hurricane-induced flooding. To evaluate change in community structure, the active zooplankton community in 2015 was compared to that observed in 1985. Shannon-Wiener and Simpson indices demonstrate that diversity of the active zooplankton community decreased during this 30-year span. In total, 31% of zooplankton species were lost, and new colonization accounts for 27% of species richness. Dominant species of all major taxonomic groupings changed. Because most zooplankton in freshwater lakes depend on dormant embryos to reestablish active populations after major disruptions, dormant embryos in the sediment "egg bank" were also quantified. Dormant cladoceran ephippia are present in bottom sediments, but dormant copepods and rotifers are missing. The existence of a dormant egg bank that is less diverse than the active community in a freshwater lake is unprecedented, and a depauperate "egg bank" would certainly impair community recovery after severe flooding from hurricanes. It is argued that a paradigm shift is needed in the ecological assessment of inland lakes in order to account for the critical role that dormant embryos (egg banks) play in freshwater zooplankton communities. Two challenges to achieving this are that 1. long-term monitoring is expensive and 2. data on dormant zooplankton are rarely available. This study provides an example of how to conduct such studies by leveraging historic data when long-term monitoring is not possible.

Embryos of an Antarctic zooplankton require anoxia for dormancy, are permeable to lipophilic chemicals, and reside in sediments containing PCBs.

Zooplankton in Antarctic maritime lakes face challenges imposed by anthropogenic chemicals. Studies on temperate species suggest that lipophilic chemicals will accumulate in dormant embryos of Antarctic zooplankton and decrease hatching success, thereby threatening centuries of accumulated genetic diversity that would increase population resilience in the face of climate change. We evaluated the potential for lakes to act as sinks for legacy pollutants in the maritime Antarctic by testing sediments for polychlorinated biphenyls (PCBs) previously identified in soil, flora and fauna of lake catchments. Direct tests of embryo permeability to chemicals are confounded by potential adhesion of chemicals to the embryo surface and limited biomass available. Therefore, in order to assess the potential for lipophilic chemicals to penetrate and passively accumulate in dormant embryos of Antarctic lacustrine zooplankton, we evaluated the effect of anoxia on post-diapause development in the calanoid copepod, Boeckella poppei, and then used chemical anoxia induced by rotenone as a reporter for permeability of these embryos to moderately lipophilic chemicals. The data presented demonstrate that embryos of B. poppei from Antarctic lake sediments will passively accumulate moderately lipophilic chemicals while lying dormant in anoxic sediments. Implications for legacy POPs in sediments of Antarctic maritime lakes are discussed.

The effects of fipronil and the photodegradation product fipronil desulfinyl on growth and gene expression in juvenile blue crabs, Callinectes sapidus, at different salinities.

Endocrine disrupting compounds (EDCs) are now widely established to be present in the environment at concentrations capable of affecting wild organisms. Although many studies have been conducted in fish, less is known about effects in invertebrates such as decapod crustaceans. Decapods are exposed to low concentrations of EDCs that may cause infertility, decreased growth, and developmental abnormalities. The objective herein was to evaluate effects of fipronil and its photodegradation product fipronil desulfinyl. Fipronil desulfinyl was detected in the eggs of the decapod Callinectes sapidus sampled off the coast of South Carolina. As such, to examine specific effects on C. sapidus exposed in early life, we exposed laboratory-reared juveniles to fipronil and fipronil desulfinyl for 96h at three nominal concentrations (0.01, 0.1, 0.5µg/l) and two different salinities (10, 30ppt). The size of individual crabs (weight, carapace width) and the expression of several genes critical to growth and reproduction were evaluated. Exposure to fipronil and fipronil desulfinyl resulted in significant size increases in all treatments compared to controls. Levels of expression for vitellogenin (Vtg), an egg yolk precursor, and the ecdysone receptor (EcR), which binds to ecdysteroids that control molting, were inversely correlated with increasing fipronil and fipronil desulfinyl concentrations. Effects on overall growth and on the expression of EcR and Vtg differ depending on the exposure salinity. The solubility of fipronil is demonstrated to decrease considerably at higher salinities. This suggests that fipronil and its photodegradation products may be more bioavailable to benthic organisms as salinity increases, as more chemical would partition to tissues. Our findings suggest that endocrine disruption is occurring through alterations to gene expression in C. sapidus populations exposed to environmental levels of fipronil, and that effects may be dependent upon the salinity at which exposure occurs.

Rotenone Decreases Hatching Success in Brine Shrimp Embryos by Blocking Development: Implications for Zooplankton Egg Banks.

While many zooplankton species recover quickly after the treatment of water resources with the piscicide, rotenone, some fail to reach pretreatment population density or, in rare cases, do not reappear at all. The variable impact of rotenone on zooplankton populations could stem from differences in the capacity of species to switch entirely to anaerobic catabolic pathways in the presence of rotenone, which blocks mitochondrial electron transport. Alternatively, variable responses among species could originate from differences in permeability of dormant life-stages to lipophilic chemicals like rotenone. The purpose of the present study was to determine the effects of rotenone on development, emergence and hatching of zooplankton embryos that lack both the anaerobic capacity to develop in the presence of rotenone and a permeability barrier to prevent the entry of rotenone during dormancy. Post-diapause embryos of the brine shrimp, Artemia franciscana, were employed as a model system, because they are permeable to lipophilic compounds when dechorionated and require aerobic conditions to support development. Early development in this species is also well characterized in the literature. Brine shrimp embryos were exposed to rotenone while development was either slowed by chilling or suspended by anoxia. Development, emergence and hatching were then observed in rotenone-free artificial seawater. The data presented demonstrate that rotenone freely diffuses across the embryonic cuticle in a matter of hours, and prevents development and emergence after brief exposures to ecologically relevant concentrations (0.025-0.5 mg L-1) of the piscicide. Neither the removal of rotenone from the environment, nor the removal of embryonic water with a hypertonic solution, are sufficient to reverse this block on development and emergence. These data indicate that rotenone could impair recruitment from egg banks for species of zooplankton that lack both an embryonic barrier to the entry of lipophilic compounds and the anaerobic capacity to develop when NADH:ubiquinone oxidoreductase activity is inhibited by rotenone.

A novel model of early development in the brine shrimp, Artemia franciscana, and its use in assessing the effects of environmental variables on development, emergence, and hatching.

The brine shrimp, Artemia (Crustacea, Anostraca), is a zooplankton that is commonly used in both basic and applied research. Unfortunately, Artemia embryos are often cultured under conditions that alter early development, and reports based on these cultures oversimplify or fail to describe morphological phenotypes. This is due in part to the lack of a comprehensive developmental model that is applicable to observations of live specimens. The objective of this study was to build and test a descriptive model of post-diapause development in Artemia franciscana using observations made with a standard dissecting microscope. The working model presented is the first to comprehensively place all known "abnormal" embryonic and naupliar phenotypes within the context of a classic hatching profile. Contrary to previous reports, embryos and nauplii with aberrant phenotypes often recover and develop normally. Oval prenauplii may emerge as normal prenauplii (E2 stage). A delay of this transition leads to incomplete hatching or direct hatching of first instar larvae with a curved thoracoabdomen. When hatching is incomplete, retained cuticular remnants are shed during the next molt, and a "normal" second instar larva is produced. By differentiating between molting events and gross embryonic patterning in live embryos, this new model facilitates fine time-scale analyses of chemical and environmental impacts on early development. A small increase in salinity within what is commonly believed to be a permissive range (20‰-35‰) produced aberrant morphology by delaying emergence without slowing development. A similar effect was observed by decreasing culture density within a range commonly applied in toxicological studies. These findings clearly demonstrate that morphological data from end-point studies are highly dependent on the time points chosen. An alternate assessment method is proposed, and the potential impact of heavy metals, hexachlorobenzene, Mirex, and cis-nonachlor detected in commercial embryos is discussed.

Rheb, an activator of target of rapamycin, in the blackback land crab, Gecarcinus lateralis: cloning and effects of molting and unweighting on expression in skeletal muscle.

Molt-induced claw muscle atrophy in decapod crustaceans facilitates exuviation and is coordinated by ecdysteroid hormones. There is a 4-fold reduction in mass accompanied by remodeling of the contractile apparatus, which is associated with an 11-fold increase in myofibrillar protein synthesis by the end of the premolt period. Loss of a walking limb or claw causes a loss of mass in the associated thoracic musculature; this unweighting atrophy occurs in intermolt and is ecdysteroid independent. Myostatin (Mstn) is a negative regulator of muscle growth in mammals; it suppresses protein synthesis, in part, by inhibiting the insulin/metazoan target of rapamycin (mTOR) signaling pathway. Signaling via mTOR activates translation by phosphorylating ribosomal S6 kinase (s6k) and 4E-binding protein 1. Rheb (Ras homolog enriched in brain), a GTP-binding protein, is a key activator of mTOR and is inhibited by Rheb-GTPase-activating protein (GAP). Akt protein kinase inactivates Rheb-GAP, thus slowing Rheb-GTPase activity and maintaining mTOR in the active state. We hypothesized that the large increase in global protein synthesis in claw muscle was due to regulation of mTOR activity by ecdysteroids, caused either directly or indirectly via Mstn. In the blackback land crab, Gecarcinus lateralis, a Mstn-like gene (Gl-Mstn) is downregulated as much as 17-fold in claw muscle during premolt and upregulated 3-fold in unweighted thoracic muscle during intermolt. Gl-Mstn expression in claw muscle is negatively correlated with hemolymph ecdysteroid level. Full-length cDNAs encoding Rheb orthologs from three crustacean species (G. lateralis, Carcinus maenas and Homarus americanus), as well as partial cDNAs encoding Akt (Gl-Akt), mTOR (Gl-mTOR) and s6k (Gl-s6k) from G. lateralis, were cloned. The effects of molting on insulin/mTOR signaling components were quantified in claw closer, weighted thoracic and unweighted thoracic muscles using quantitative polymerase chain reaction. Gl-Rheb mRNA levels increased 3.4-fold and 3.9-fold during premolt in claw muscles from animals induced to molt by eyestalk ablation (ESA) and multiple leg autotomy (MLA), respectively, and mRNA levels were positively correlated with hemolymph ecdysteroids. There was little or no effect of molting on Gl-Rheb expression in weighted thoracic muscle and no correlation of Gl-Rheb mRNA with ecdysteroid titer. There were significant changes in Gl-Akt, Gl-mTOR and Gl-s6k expression with molt stage. These changes were transient and were not correlated with hemolymph ecdysteroids. The two muscles differed in terms of the relationship between Gl-Rheb and Gl-Mstn expression. In thoracic muscle, Gl-Rheb mRNA was positively correlated with Gl-Mstn mRNA in both ESA and MLA animals. By contrast, Gl-Rheb mRNA in claw muscle was negatively correlated with Gl-Mstn mRNA in ESA animals, and no correlation was observed in MLA animals. Unweighting increased Gl-Rheb expression in thoracic muscle at all molt stages; the greatest difference (2.2-fold) was observed in intermolt animals. There was also a 1.3-fold increase in Gl-s6k mRNA level in unweighted thoracic muscle. These data indicate that the mTOR pathway is upregulated in atrophic muscles. Gl-Rheb, in particular, appears to play a role in the molt-induced increase in protein synthesis in the claw muscle.

Metabolic restructuring during energy-limited states: insights from Artemia franciscana embryos and other animals.

Many life history stages of animals that experience environmental insults enter developmental arrested states that are characterized by reduced cellular proliferation, with or without a concurrent reduction in overall metabolism. In the case of the most profound metabolic arrest reported in invertebrates, i.e., anaerobic quiescence in Artemia franciscana embryos, acidification of the intracellular milieu is a major factor governing catabolic and anabolic downregulation. Release of ions from intracellular compartments is the source for approximately 50% of the proton equivalents needed for the 1.5 unit acidification that is observed. Recovery from the metabolic arrest requires re-sequestration of the protons with a vacuolar-type ATPase (V-ATPase). The remarkable facet of this mechanism is the ability of embryonic cells to survive the dissipation of intracellular ion gradients. Across many diapause-like states, the metabolic reduction and subsequent matching of energy demand is accomplished by shifting energy metabolism from oxidative phosphorylation to aerobic glycolysis. Molecular pathways that are activated to induce these resilient hypometabolic states include stimulation of the AMP-activated protein kinase (AMPK) and insulin signaling via suite of daf (dauer formation) genes for diapause-like states in nematodes and insects. Contributing factors for other metabolically depressed states involve hypoxia-inducible factor-1 and downregulation of the pyruvate dehydrogenase complex. Metabolic similarities between natural states of stasis and some cancer phenotypes are noteworthy. Reduction of flux through oxidative phosphorylation helps prevent cell death in certain cancer types, similar to the way it increases viability of dauer stages in Caenorhabditis elegans. Mechanisms that underlie natural stasis are being used to pre-condition mammalian cells prior to cell biostabilization and storage.

Myostatin from the American lobster, Homarus americanus: Cloning and effects of molting on expression in skeletal muscles.

A cDNA encoding a myostatin (Mstn)-like gene from an astacuran crustacean, Homarus americanus, was cloned and characterized. Mstn inhibits skeletal muscle growth in vertebrates and may play a role in crustacean muscle as a suppressor of protein synthesis. Sequence analysis and three-dimensional modeling of the Ha-Mstn protein predicted a high degree of conservation with vertebrate and other invertebrate myostatins. Qualitative polymerase chain reaction (PCR) demonstrated ubiquitous expression of transcript in all tissues, including skeletal muscles. Quantitative PCR analysis was used to determine the effects of natural molting and eyestalk ablation (ESA) on Ha-Mstn expression in the cutter claw (CT) and crusher claw (CR) closer muscles and deep abdominal (DA) muscle. In intermolt lobsters, the Ha-Mstn mRNA level in the DA muscle was significantly lower than the mRNA levels in the CT and CR muscles. Spontaneous molting decreased Ha-Mstn mRNA during premolt, with the CR muscle, which is composed of slow-twitch (S1) fibers, responding preferentially (82% decrease) to the atrophic signal compared to fast fibers in CT (51% decrease) and DA (69% decrease) muscles. However, acute increases in circulating ecdysteroids caused by ESA had no effect on Ha-Mstn mRNA levels in the three muscles. These data indicate that the transcription of Ha-Mstn is differentially regulated during the natural molt cycle and it is an important regulator of protein turnover in molt-induced claw muscle atrophy.

Conserved role of cyclic nucleotides in the regulation of ecdysteroidogenesis by the crustacean molting gland.

Molting processes in crustaceans are regulated by ecdysteroids produced in the molting gland (Y-organ), and molting is indirectly controlled by circulating factors that inhibit the production of these polyhydroxylated steroids. Two of these regulatory factors are the neuropeptides molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). CHH appears to inhibit ecdysteroidogenesis in the Y-organ through the activation of a receptor guanylyl cyclase. The signaling pathway activated by MIH, however, remains a subject of controversy. It is clear that neuropeptides inhibit ecdysteroidogenesis by simultaneously suppressing ecdysteroid biosynthetic processes, protein synthesis, and uptake of high density lipoproteins. Data demonstrate that cAMP is the primary regulator of critical catabolic, anabolic, and transport processes, which ultimately support the capacity for ecdysteroid production by the Y-organ. While cAMP also regulates acute ecdysteroidogenesis to some extent, data indicate that cGMP is the primary signaling molecule responsible for acute inhibition by neuropeptides. It is clear that the regulatory roles filled by cAMP and cGMP are conserved among decapod crustaceans. It is unknown if these complementary second messengers are linked in a single signaling pathway or are components of independent pathways activated by different factors present in extracts of eyestalk ganglia.

Expression of alternatively spliced transcripts for a myostatin-like protein in the blackback land crab, Gecarcinus lateralis.

Three complete cDNAs for the first myostatin-like gene identified in a crustacean species were cloned from the land crab, Gecarcinus lateralis. Sequence analysis demonstrates a high degree of conservation with myostatin orthologs from vertebrates. The furin cleavage site is identical to that of human myostatin, and all nine cysteines critical to the structure/function of mature myostatin peptides are conserved. Message levels for transcripts encoding the complete crustacean preproprotein were highest in skeletal muscle and heart. Lower levels of expression were observed in nervous tissue, gill, gonad, and hepatopancreas. This expansive distribution is similar to that observed for teleost myostatin, vertebrate GDF-11, and amphioxus GDF8/11, and indicates a potentially broad functional repertoire for the land crab ortholog. In addition to one cDNA encoding a complete preproprotein, two cDNAs encoding C-terminal truncated proteins lacking a mature peptide domain were identified. Expression of these truncated splice variants was restricted to skeletal muscle and heart. Myostatin is a potent negative regulator of muscle mass in mammals, and strong expression of this TGF-beta factor in skeletal muscle during intermolt indicates that a myostatin-like gene product could regulate muscle mass in crustaceans when growth is physically restricted by a calcified exoskeleton.

Energizing an invertebrate embryo: bafilomycin-dependent respiration and the metabolic cost of proton pumping by the V-ATPase.

We examine herein the contribution of V-ATPase activity to the energy budget of aerobically developing embryos of Artemia franciscana and discuss the results in the context of quiescence under anoxia. (31)P-NMR analysis indicates that intracellular pH and NTP levels are unaffected by acute incubation of dechorionated embryos with the V-ATPase inhibitor, bafilomycin A(1). Bafilomycin A(1) also has no significant effect on oxygen consumption by isolated mitochondria. Taken together, these data indicate that bafilomycin does not affect energy-producing pathways in the developing embryo. However, the V-ATPase inhibitor exhibits a concentration-dependent inhibition of oxygen consumption in aerobic embryos. A conservative analysis of respirometric data indicates that proton pumping by the V-ATPase, and processes immediately dependent on this activity, constitutes approximately 31% of the aerobic energy budget of the preemergent embryo. Given the complete absence of detectable Na(+)K(+)-ATPase activity during the first hours of aerobic development, it is plausible that the V-ATPase is performing a role in both the acidification of intracellular compartments and the energization of plasma membranes. Importantly, the high metabolic cost associated with maintaining these diverse proton gradients requires that V-ATPase activity be downregulated under anoxia in order to attain the almost complete metabolic depression observed in the quiescent embryo.

Molt-inhibiting hormone from the tropical land crab, Gecarcinus lateralis: cloning, tissue expression, and expression of biologically active recombinant peptide in yeast.

Molt-inhibiting hormone (MIH), a member of the crustacean hyperglycemic neuropeptide hormone family, inhibits ecdysteroidogenesis in the molting gland or Y-organ (YO). A cDNA encoding MIH of the land crab (Gel-MIH) was cloned from eyestalk ganglia (EG) by a combination of reverse transcriptase polymerase chain reaction (RT-PCR) and 3'- and 5'-rapid amplification of cDNA ends (RACE). The cDNA (1.4 kb) encoded MIH prohormone containing a 35 amino acid signal peptide and a 78 amino acid mature peptide. The mature peptide had the six cysteines, one glycine, two arginines, one aspartate, one phenylalanine, and one asparagine in identical positions in the highly conserved sequence characteristic of other crustacean MIHs. Gel-MIH was expressed only in the EG, as determined by RT-PCR; it was not detected in Y-organ, heart, integument, gill, testis, ovary, hepatopancreas, thoracic ganglion, or skeletal muscle. A cDNA encoding the mature peptide was used to express recombinant MIH (rMIH) using a yeast (Pichia pastoris) expression system. Two constructs were designed to yield either a mature MIH fusion protein with a c-myc epitope and histidine (His) tag at the carboxyl terminus or an untagged mature protein without the c-myc and His sequences. Immunoreactive peptides were detected in Western blots of the cell culture media with both MIH constructs, indicating secretion of the processed rMIH into the medium. Culture media containing the untagged mature peptide significantly inhibited ecdysteroid secretion by YOs from land crab and green crab (Carcinus maenas) cultured in vitro, indicating that the Gel-rMIH was biologically active.

V-ATPase expression during development of Artemia franciscana embryos: potential role for proton gradients in anoxia signaling.

Under anoxia, 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 ;generalist-type' 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 V-ATPase inhibitor bafilomycin A1, which halts embryonic development. Importantly, net proton flux from V-ATPase-acidified compartments to the surrounding cytoplasm is likely under anoxia and may significantly contribute to the enigmatic acidification critical to quiescence.

V-ATPase inhibition prevents recovery from anoxia in Artemia franciscana embryos: quiescence signaling through dissipation of proton gradients.

The metabolic downregulation critical for long-term survival of Artemia franciscana embryos under anoxia is mediated, in part, by a progressive intracellular acidification. However, very little is known about the mechanisms responsible for the pH transitions associated with exposure to, and recovery from, oxygen deprivation. In the present study, we demonstrate with 31P-NMR that incubation of intact embryos with the V-ATPase inhibitor bafilomycin A1 severely limits intracellular alkalinization during recovery from anoxia without affecting the restoration of cellular nucleotide triphosphate levels. Based on these data, it appears that oxidative phosphorylation and ATP resynthesis can only account for the first 0.3 pH unit alkalinization observed during aerobic recovery from the 1 pH unit acidification produced during 1 h of anoxia. The additional 0.7 pH unit increase requires proton pumping by the V-ATPase. Aerobic incubation with bafilomycin also suggests that V-ATPase inhibition alone is not enough to induce an acute dissipation of proton gradients under anoxia. In intact embryos, the dissipation of proton gradients and uncoupling of oxidative phosphorylation with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) leads to an intracellular acidification similar to that seen after 1 h of anoxia. Subsequent exposure to anoxia, in the continued presence of CCCP, yields little additional acidification, suggesting that proton gradients are normally dissipated under anoxia. When combined with protons generated from net ATP hydrolysis, these data show that the dissipation of proton chemical gradients is sufficient to account for the reversible acidification associated with quiescence in these embryos.