The total and mitochondrial lipidome of Artemia franciscana encysted embryos

Encysted embryos (cysts) of the crustacean Artemia franciscana exhibit enormous tolerance to adverse conditions encompassing high doses of radiation, years of anoxia, desiccation and extreme salinity. So far, several mechanisms have been proposed to contribute to this extremophilia, however, none were sought in the lipid profile of the cysts. Here in, we used high resolution shotgun lipidomics suited for detailed quantitation and analysis of lipids in uncharacterized biological membranes and samples and assembled the total, mitochondrial and mitoplastic lipidome of Artemia franciscana cysts. Overall, we identified and quantitated 1098 lipid species dispersed among 22 different classes and subclasses. Regarding the mitochondrial lipidome, most lipid classes exhibited little differences from those reported in other animals, however, Artemia mitochondria harboured much less phosphatidylethanolamine, plasmenylethanolamines and ceramides than mitochondria of other species, some of which by two orders of magnitude. Alternatively, Artemia mitochondria exhibited much higher levels of phosphatidylglycerols and phosphatidylserines. The identification and quantitation of the total and mitochondrial lipidome of the cysts may help in the elucidation of actionable extremophilia-affording proteins, such as the ‘late embryogenesis abundant’ proteins, which are known to interact with lipid membranes.     

Long-term anoxia in encysted embryos of the crustacean, Artemia franciscana: viability, ultrastructure, and stress proteins

Cells of encysted embryos of Artemia franciscana, the brine shrimp, are among the most resistant of all animal cells to extremes of environmental stress. We focus here on their ability to survive continuous anoxia for periods of years, during which their metabolic rate is undetectable. We asked whether their impressive tolerance was reflected in changes at the ultrastructural level. The ultrastructure of encysted embryos previously experiencing 38 days and 3.3 years of anoxia was compared with those not undergoing anoxia (controls). Rough endoplasmic reticulum was abundant in anoxic embryos, in spite of the absence of protein biosynthesis in their cells. Other cytoplasmic changes had occurred in the anoxic cells, but overall their structure was remarkably intact, in view of their 3 years of continuous anoxia. A major difference was the presence of abundant electron-dense granules in the nuclei of anoxic embryos; these were present but rare in nuclei of controls. Biochemical fractionation and Western immunoblotting confirmed previous observations that substantial amounts of the small heat shock/alpha-crystallin protein (p26) translocated into nuclei of anoxic embryos. We have no evidence that the dense granules contain this protein, but that remains a possibility. In contrast, and contrary to expectation, proteins of the hsp70 and 90 families did not undergo anoxia-induced nuclear translocation, an unusual result since such translocations have been widely observed in cells from a variety of organisms.     

Stress-related proteins compared in diapause and in activated, anoxic encysted embryos of the animal extremophile, Artemia franciscana

Previous work indicated similarities between diapause and anoxic quiescence in encysted embryos (cysts) of the brine shrimp Artemia franciscana. That possibility was examined further in the present study through an immunochemical study of the following stress-related proteins in low speed supernatants and pellets: hsc70, artemin, p26, hsp21, LEA Group 1 protein and p8. Changes in the amounts and locations of these proteins occurred during the initial period after release of diapause cysts from females, and after activated (diapause-terminated) cysts were made anoxic. However, with the passage of incubation time the patterns seen in both kinds of cysts were more similar than different, lending further support to the possibility that activated anoxic embryos retain many of the mechanisms operative in the previous diapause condition.     

Activation of an AMP-activated protein kinase is involved in post-diapause development of Artemia franciscana encysted embryos

Background  

Cysts of Artemia can remain in a dormant state for long periods with a very low metabolic rate, and only resume their development with the approach of favorable conditions. The post-diapause development is a very complicated process involving a variety of metabolic and biochemical events. However, the intrinsic mechanisms that regulate this process are unclear.     

Intracellular localization of group 3 LEA proteins in embryos of Artemia franciscana

Late embryogenesis abundant (LEA) proteins are accumulated by anhydrobiotic organisms in response to desiccation and improve survivorship during water stress. In this study we provide the first direct evidence for the subcellular localizations of AfrLEA2 and AfrLEA3m (and its subforms) in anhydrobiotic embryos of Artemia franciscana. Immunohistochemistry shows AfrLEA2 to reside in the cytoplasm and nucleus, and the four AfrLEA3m proteins to be localized to the mitochondrion. Cellular locations are supported by Western blots of mitochondrial, nuclear and cytoplasmic fractions. The presence of LEA proteins in multiple subcellular compartments of A. franciscana embryos suggests the need to protect biological structures in many areas of a cell in order for an organism to survive desiccation stress, and may explain in part why a multitude of different LEA proteins are expressed by a single organism.     

Characteristics and intracellular distribution of messengerlike RNA in encysted embryos of Artemia salina

Homogenates of dormant cysts of Artemia salina were fractionated by differential centrifugation. RNA was prepared from the various fractions and tested for stimulatory activity in a [¹⁴C]leucine incorporating Escherichia coli system. The highest specific activity was found in the RNA extracted from a cytoplasmic fraction sedimenting at 15,000 g. Some activity was associated with the soluble and crude ribosomal fractions, while the RNA extracted from the crude nuclear fraction was less active.The 15,000 g sediment was purified by centrifugation in a sucrose density gradient. The active material formed a characteristic, colored band at a buoyant density of about 1.17 g/ml. The banding fraction was mainly composed of endoplasmic vesicles and mitochondria. The specific activity of the extracted RNA was further increased when the 15,000 g sediment was treated with buffered 20–100 mM EDTA (with or without 0.1% Triton X-100) before banding.Sedimentation analysis of the active RNA from the purified 15,000 g fractions revealed three distinct absorption peaks at 28 S, 18 S, and 16 S, apparently representing cytoplasmic and mitochondrial rRNA. The 28 S and 18 S peaks were reduced by EDTA treatment, but only to a certain limit. By gel electrophoresis a number of additional components were resolved, including 4 S and 5 S RNA. The template activity showed a heterodisperse distribution with a maximum at 17–20 S, not correlated with the 16 S peak. Isolated 18 S and 28 S rRNA had very low activity.The experiments suggest that in Artemia cysts an appreciable amount of messengerlike RNA is associated with mitochondria and/or endoplasmic vesicles carrying ribosomal monomers.     

Group 1 LEA proteins contribute to the desiccation and freeze tolerance of Artemia franciscana embryos during diapause

Water loss either by desiccation or freezing causes multiple forms of cellular damage. The encysted embryos (cysts) of the crustacean Artemia franciscana have several molecular mechanisms to enable anhydrobiosis—life without water—during diapause. To better understand how cysts survive reduced hydration, group 1 late embryogenesis abundant (LEA) proteins, hydrophilic unstructured proteins that accumulate in the stress-tolerant cysts of A. franciscana, were knocked down using RNA interference (RNAi). Embryos lacking group 1 LEA proteins showed significantly lower survival than control embryos after desiccation and freezing, or freezing alone, demonstrating a role for group 1 LEA proteins in A. franciscana tolerance of low water conditions. In contrast, regardless of group 1 LEA protein presence, cysts responded similarly to hydrogen peroxide (H₂O₂) exposure, indicating little to no function for these proteins in diapause termination. This is the first in vivo study of group 1 LEA proteins in an animal and it contributes to the fundamental understanding of these proteins. Knowing how LEA proteins protect A. franciscana cysts from desiccation and freezing may have applied significance in aquaculture, where Artemia is an important feed source, and in the cryopreservation of cells for therapeutic applications.     

Molecular chaperones, stress resistance and development in Artemia franciscana

Embryos of the brine shrimp, Artemia franciscana, either develop directly into swimming larvae or are released from females as encysted gastrulae (cysts) which enter diapause, a reversible state of dormancy. Metabolic activity in diapause cysts is very low and these embryos are remarkably resistant to physiological stresses. Encysting embryos, but not those undergoing uninterrupted development, synthesize large amounts of two proteins, namely p26 and artemin. Cloning and sequencing demonstrated p26 is a small heat shock/alpha-crystallin protein while artemin has structural similarity to ferritin. p26 exhibits molecular chaperone activity in vitro, moves reversibly into nuclei during stress and confers thermotolerance on transformed organisms, suggesting critical roles in cyst development. The function of artemin is unknown. Encysted Artemia also contain an abundance of trehalose, a disaccharide capable of protecting embryos. Artemia represent a novel experimental system where the developmental functions of small heat shock/alpha-crystallin proteins and other stress response elements can be explored.     

Evidence of a store of informational RNA in encysted embryos of Artemia salina

RNA prepared from dormant cysts and developmental stages of the brine shrimp Artemia salina stimulated the incorporation of ¹⁴C-leucine into polypeptide by a cell-free Escherichia coli system. Preparations from cysts were about as active as those from hatching embryos or nauplii. When analysed by density gradient centrifugation the activity of cyst RNA showed a heterodisperse distribution, not quantitatively related to the absorbance profile. These results and evidence from similar experiments with crude ribosome preparations indicated that the contribution of 18S and 28S ribosomal RNA to the template-like activity was fairly limited. The experiments suggest that RNA with latent messenger activity is present in Artemia cysts during the resting stage.     

Aerobic heat shock activates trehalose synthesis in embryos of Artemia franciscana.

Encysted embryos (cysts) of the brine shrimp, Artemia franciscana, contain large amounts of trehalose which they use as a major substrate for energy metabolism and biosynthesis for development under aerobic conditions at 25 degrees C. When cysts are placed at 42 degrees C (heat shock) these pathways stop, and the cysts re-synthesize the trehalose that was utilized during the previous incubation at 25 degrees C. Glycogen and glycerol, produced from trehalose at 25 degrees C, appear to be substrates for trehalose synthesis during heat shock. Anoxia prevents trehalose synthesis in cysts undergoing heat shock. These results are consistent with the view that trehalose may play a protective role in cells exposed to heat shock, and other environmental insults, in addition to being a storage form of energy and organic carbon for development.     

Quantification of cellular protein expression and molecular features of group 3 LEA proteins from embryos of Artemia franciscana

Late embryogenesis abundant (LEA) proteins are highly hydrophilic, low complexity proteins whose expression has been correlated with desiccation tolerance in anhydrobiotic organisms. Here, we report the identification of three new mitochondrial LEA proteins in anhydrobiotic embryos of Artemia franciscana, AfrLEA3m_47, AfrLEA3m_43, and AfrLEA3m_29. These new isoforms are recognized by antibody raised against recombinant AfrLEA3m, the original mitochondrial-targeted LEA protein previously reported from these embryos; mass spectrometry confirms all four proteins share sequence similarity. The corresponding messenger RNA (mRNA) species for the four proteins are readily amplified from total complementary DNA (cDNA) prepared from embryos. cDNA sequences of the four mRNAs are quite similar, but each has a stretch of sequence that is absent in at least one of the others, plus multiple single base pair differences. We conclude that all four mitochondrial LEA proteins are products of independent genes. Each possesses a mitochondrial targeting sequence, and indeed Western blots performed on extracts of isolated mitochondria clearly detect all four isoforms. Based on mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis migration, the cytoplasmic-localized AfrLEA2 exists primarily as a homodimer in A. franciscana. Quantification of protein expression for AfrLEA2, AfrLEA3m, AfrLEA3m_43, and AfrLEA3m_29 as a function of development shows that cellular concentrations are highest in diapause embryos and decrease during development to low levels in desiccation-intolerant nauplius larvae. When adjustment is made for mitochondria matrix volume, the effective concentrations of cytoplasmic versus mitochondrial group 3 LEA proteins are similar in vivo, and the values provide guidance for the design of in vitro functional studies with these proteins.     

Characterization of ATP-dependent proteolysis in embryos of the brine shrimp, Artemia franciscana

Under anoxia, embryos of Artemia franciscana enter a state of quiescence. During this time protein synthesis is depressed, and continued degradation of proteins could jeopardize the ability to recover from quiescence upon return to favorable conditions. In this study, we developed an assay for monitoring ATP/ubiquitin-dependent proteolysis in order to establish the presence of this degradation mechanism in A. franciscana embryos, and to describe some characteristics that may regulate its function during anoxia-induced quiescence. For lysates experimentally depleted of adenylates, supplementation with ATP and ubiquitin stimulated protein degradation rates by 92 ± 17% (mean ± SE) compared to control rates. The stimulation by ATP was maximal at concentrations ≥11 μmol · l⁻¹. In the presence of ATP and ubiquitin, ubiquitin-conjugated proteins were produced by lysates during the course of the 4-h assays, as detected by Western blotting. Acute acidification of lysates to values approximating the intracellular pH observed under anoxia completely inhibited ATP/ubiquitin-dependent proteolysis. Depressed degradation was also observed under conditions where net ATP hydrolysis occurred. These results suggest that ATP/ubiquitin-dependent proteolysis is markedly inhibited under cellular conditions promoted by anoxia. Inhibition of proteolysis during quiescence may be one critical factor that increases macromolecular stability, which may ultimately govern the duration of embryo survival under anoxia. Accepted: 2 November 1999     

Respiration of Artemia franciscana embryos after continuous anoxia over 1-year period

Encysted embryos of the brine shrimp, Artemia franciscana, exhibit extraordinary longevity when exposed to continuous anoxia. To explore the metabolic basis of this ability, the post-anoxic respiration of embryos exposed to anoxia for periods exceeding 1 year was measured. Since anoxic metabolism might result in the accumulation of metabolic end products, an O₂ debt would be expected. Contrary to that expectation, post-anoxic embryos exhibited a marked depression in respiration rate whether embryos were hydrated under anoxic conditions or were exposed to a previous aerobic incubation and then placed under anoxia. These results, and those of previous studies, suggest that extended anoxia may bring the metabolism of these embryos to a reversible standstill.     

Extracellular matrix peptides of Artemia cyst shell participate in protecting encysted embryos from extreme environments

Background

Many species of the brine shrimp Artemia are found in various severe environments in many parts of the world where extreme salinity, high UV radiation levels, high pH, anoxia, large temperature fluctuations, and intermittent dry conditions are often recorded. To withstand adverse environments, Artemia undergoes an oviparous developmental pathway to release cysts whereas, under favorable conditions, swimming nauplius larvae are formed directly via an ovoviviparous pathway. In the former case these cysts have an extraordinary ability to keep the embryos protected from the harsh environment for long periods. This is achieved through the protection by a complex out-wrapping cyst shell. However, the formation and function of the cyst shell is complex; the details remain largely unclear.

Principal Finding

A shell gland-specific gene (SGEG2) was cloned and identified from a suppression subtractive hybridization library. Western blot analysis showed that SGEG2 presumably requires post-translational proteolysis in order to be processed into two mature peptides (SGEG2a and 2b). The three matrix peptides (SGEG1 reported previously, 2a, and 2b) were found to distribute throughout the cyst shell. The results of gene knockdown by RNAi and subsequent resistance to environmental stresses assays indicated that these matrix peptides are required for cyst shell formation and are involved in protecting the encysted embryos from environmental stress.

Conclusions/Significance

This study revealed that extracellular matrix peptides participate in protecting embryos from extreme salinity, UV radiation, large temperature fluctuations and dry environments, thereby facilitating their survival. The cyst shell provides an excellent opportunity to link the ecological setting of an organism to the underlying physiological and biochemical processes enabling its survival. The cyst shell material has also a high potential to become an excellent new biomaterial with a high number of prospective uses due, specifically, to such biological characteristics.     

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.     

Mechanisms associated with cellular desiccation tolerance of Artemia encysted embryos from locations around the world

Using differential scanning calorimetry we demonstrated the presence of biological glasses and measured the glass transition temperatures (Tg) in dry encysted gastrula embryos (cysts) of the brine shrimp, Artemia, from eleven different locations, two of which provided cysts from parthenogenetic animals. Values for Tg were highest, by far, in Artemia franciscana cysts from the Mekong Delta, Vietnam (VN), these cysts having been produced from previous sequential inoculations into growth ponds of cysts from the San Francisco Bay, California, USA. Tg values for three groups of A. franciscana cysts were significantly higher than those of other cysts (except those of Artemia persimilis) studied here, as well as all other desiccation-tolerant animal systems studied to date. We also measured three stress proteins (hsc70, artemin and p26) in all these cysts as well as the total alcohol soluble carbohydrates (ASC), about 90% of which is the disaccharide trehalose, a known component of biological glasses. We interpret the results in terms of mechanisms involved with desiccation tolerance and, to some extent, with thermal conditions at the sites of cyst collection.     

Maternal effects on encystment in crosses between two geographic strains of Artemia franciscana

Maternal effects can have environmental or genetic causes. A method that can be used to demonstrate the genetic basis of a maternal effect is to look for grandfather effects in a backcross following reciprocal crosses. The absence of a grandfather effect would exclude a chromosomal basis for the maternal effect when the male sex is heterogametic (XX-XY sex determination system). However, in organisms in which the female is heterogametic (ZW-ZZ sex determination system), the absence of a grandfather effect does not rule out a chromosomal basis of the maternal effect, since the genes responsible for that effect can be located in the W chromosome, which is transmitted matrilineally. Conversely, the absence of a grandfather effect would point to a W-chromosome basis for the trait, provided that a maternal effect has been previously demonstrated. Distinguishing between W-located and autosome or Z-located maternal effects is important to understand the evolutionary dynamics of a trait. Here we report on a study of the chromosomal basis of maternal effects on two life-history traits related to encystment in the branchiopod crustacean Artemia franciscana, in which females are heterogametic. We performed crosses of two populations that differ in the number of cysts they produce. The proportion of encysted broods showed a maternal effect and was not affected by the grandfather's genotype, pointing to a W-chromosome basis. The average number of encysted offspring per brood showed a strong paternal effect and also a slight maternal effect. This trait also showed a grandfather effect, which suggests that the geographical variation has an autosomal or Z-chromosomal basis.