Mitochondrial physiology of diapausing and developing embryos of the annual killifish Austrofundulus limnaeus: implications for extreme anoxia tolerance

Diapausing embryos of the annual killifish Austrofundulus limnaeus have the highest reported anoxia tolerance of any vertebrate and previous studies indicate modified mitochondrial physiology likely supports anoxic metabolism. Functional mitochondria isolated from diapausing and developing embryos of the annual killifish exhibited VO₂, respiratory control ratios (RCR), and P:O ratios consistent with those obtained from other ectothermic vertebrate species. Reduced oxygen consumption associated with dormancy in whole animal respiration rates are correlated with maximal respiration rates of mitochondria isolated from diapausing versus developing embryos. P:O ratios for developing embryos were similar to those obtained from adult liver, but were diminished in mitochondria from diapausing embryos suggesting decreased oxidative efficiency. Proton leak in adult liver corresponded with that of developing embryos but was elevated in mitochondria isolated from diapausing embryos. In metabolically suppressed diapause II embryos, over 95% of the mitochondrial oxygen consumption is accounted for by proton leak across the inner mitochondrial membrane. Decreased activity of mitochondrial respiratory chain complexes correlates with diminished oxidative capacity of isolated mitochondria, especially during diapause. Respiratory complexes exhibited suppressed activity in mitochondria with the ATP synthase exhibiting the greatest inhibition during diapause II. Mitochondria isolated from diapause II embryos are not poised to produce ATP, but rather to shuttle carbon and electrons through the Kreb’s cycle while minimizing the generation of a proton motive force. This particular mitochondrial physiology is likely a mechanism to avoid production of reactive oxygen species during large-scale changes in flux through oxidative phosphorylation pathways associated with metabolic transitions into and out of dormancy and anoxia.     

Characterization of sub-nuclear changes in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> embryos exposed to brief,intermediate and long-term anoxia to analyze anoxia-induced cell cycle arrest

Background  

The soil nematode C. elegans survives oxygen-deprived conditions (anoxia; <.001 kPa O₂) by entering into a state of suspended animation in which cell cycle progression reversibly arrests. The majority of blastomeres of embryos exposed to anoxia arrest at interphase, prophase and metaphase. The spindle checkpoint proteins SAN-1 and MDF-2 are required for embryos to survive 24 hours of anoxia. To further investigate the mechanism of cell-cycle arrest we examined and compared sub-nuclear changes such as chromatin localization pattern, post-translational modification of histone H3, spindle microtubules, and localization of the spindle checkpoint protein SAN-1 with respect to various anoxia exposure time points. To ensure analysis of embryos exposed to anoxia and not post-anoxic recovery we fixed all embryos in an anoxia glove box chamber.     

Heat dissipation during long-term anoxia inArtemia franciscana embryos: identification and fate of metabolic fuels

Summary Microcalorimetric measurements of brine shrimp embryos during 6 days of anoxia indicated that heat dissipation was rapidly suppressed to 2.7% of control (aerobic) values over the first 9 h. Energy flow continued to decline slowly to 31 W·g dry mass^-1 (0.4% of control) during the subsequent 5.5 days. Within 2 h after returning anoxic embryos to aerobic conditions, heat dissipation rose to 77% of control rates. The calorimetric/respirometric (CR) ratio across this 2-h recovery period increased steadily from-226 to-346 kJ·mol O ₂ ^-1 . Prior to the anoxic exposures, hydrated embryos were incubated aerobically for 10 h to insure full initiation of carbohydrate metabolism (CR ratio=-484 kJ·mol O ₂ ^-1 ). During the 6-day asymptotic approach to a nearly ametabolic state, trehalose and glycogen levels declined 18% and 13%, respectively. The majority of this utilization occurred within the first three days. Thermochemical calculations showed that carbohydrate catabolism accounted for 84% of the total heat dissipation measured over the 6-day anoxic bout; only 3% of the heat could be explained by the catabolism of diguanosine tetraphosphate (Gp₄G). Analyses of embryo extracts by high performance liquid chromatography indicated that multiple acid end products were accumulated. Lactate and propionate reached 4.5 mM and 1.0 mM, respectively, but these compounds did not account quantitatively for the amount of carbohydrate utilized. However, the largest chromatographic peak that accumulated under anoxia has not been successfully identified. Fumarate and pyruvate levels decreased as anoxia proceeded. Thus, a perceptible energy flow inArtemia franciscana embryos still remained after 6 days of anoxia. While an ametabolic state may be reached with time, the length of this prolonged transition into anaerobic dormancy has not been appreciated before.     

Supply and partitioning of assimilates to roots of Medicago sativa L. and Lotus corniculatus L. under anoxia

Abstract A current explanation of the mechanism of flooding injury to roots suggests that oxygen deficiency depresses the supply of respirable carbohydrates sufficiently to inhibit fermentation. However, even though it has been shown that phloem transport of assimilate is sharply reduced to anaerobic roots, inhibition of assimilate metabolism has also been suggested to be an important factor. This study examines these hypotheses by relating assimilate supply and metabolic activity in anoxic roots of alfalfa (Medicago sativa L.), a flood-intolerant species, and birdsfoot trefoil (Lotus corniculatus L.), a flood-tolerant plant. Roots were made anoxic (severe O₂ deficiency) for 2, 4 or 6 d and shoots were labelled with ¹⁴CO₂. Assimilate transport to the roots and metabolism to structural components were significantly decreased in both species in response to anoxia. Trefoil exhibited significantly greater ¹⁴C incorporation into the residue fraction at 4 d anoxia than did alfalfa, and this was consistent with the greater flooding tolerance of trefoil. When assimilate supply to O₂-deficient roots was decreased by shoot shading, shoot fresh weight was reduced by both anoxia and light treatments. Root-soluble sugars were significantly decreased by shading but were greatly increased in response to anoxia. Root starch concentration also increased under anoxia. Root K⁺ concentration was reduced by anoxia only. The energy status (ATP/ADP) of roots was significantly decreased by shading; however, anoxia reduced the energy status only in unshaded plants. The data indicate that carbohydrate supply to anaerobic roots does not appear to be a limiting factor in the metabolic response of alfalfa roots. Alternatively, metabolism of assimilate in anoxic roots may be an important determinant of survival.     

Anoxic survival of the Pacific hagfish (<Emphasis Type="Italic">Eptatretus stoutii</Emphasis>)

It is not known how the Pacific hagfish (Eptatretus stoutii) can survive extended periods of anoxia. The present study used two experimental approaches to examine energy use during and following anoxic exposure periods of different durations (6, 24 and 36 h). By measuring oxygen consumption prior to anoxic exposure, we detected a circadian rhythm, with hagfish being active during night and showing a minimum routine oxygen consumption (RMR) during the daytime. By measuring the excess post-anoxic oxygen consumption (EPAOC) after 6 and 24 h it was possible to mathematically account for RMR being maintained even though heme stores of oxygen would have been depleted by the animal’s metabolism during the first hours of anoxia. However, EPAOC after 36 h of anoxia could not account for RMR being maintained. Measurements of tissue glycogen disappearance and lactate appearance during anoxia showed that the degree of glycolysis and the timing of its activation varied among tissues. Yet, neither measurement could account for the RMR being maintained during even the 6-h anoxic period. Therefore, two independent analyses of the metabolic responses of hagfish to anoxia exposure suggest that hagfish utilize metabolic rate suppression as part of the strategy for longer-term anoxia survival.     

Metabolic adaptation to prolonged anoxia in leaves of American cranberry (Vaccinium macrocarpon)

The indigenous North American Cranberry ( Vaccinium macrocarpon ), when cultivated in specially constructed cranberry bogs, is normally flooded in winter to prevent frost injury. This protection under ice can give rise to prolonged periods of anoxia, which depending on the state of the vines and environmental conditions, can cause severe oxygen-deprivation injury. An experimental study of the tolerance of cranberry vines to controlled total anoxia reveals that mature dark-green perennating leaves with high carbohydrate levels are able to survive prolonged periods of total oxygen-deprivation while younger newly formed leaves are readily damaged. During the anoxic treatment the mature leaves exhibit a marked downregulation of metabolism. Carbohydrate consumption and energy metabolism stabilize at low levels soon after the switch from aerobic to anaerobic pathways. Pathways such as TCA cycle or photosynthesis, which are non-operating during the anoxia treatment, are severely affected but still measurable after 28 days anoxia. In the post-anoxic period the perennating leaves rapidly re-establish their capacity for aerobic respiration and photosynthesis.     

Regulation of tail muscle arginine kinase by reversible phosphorylation in an anoxia-tolerant crayfish

Freshwater crayfish, Orconectes virilis, can experience periodic exposures to hypoxia or anoxia due to low water flow (in summer) or ice cover (in winter) in their natural habitat. Hypoxia/anoxia disrupts energy metabolism and triggers mechanisms that to support ATP levels while often also suppressing ATP use. Arginine kinase (AK) (E.C. 2.7.3.3) is a crucial enzyme involved in energy metabolism in muscle, gating the use of phosphagen stores to buffer ATP levels. The present study investigated AK from tail muscle of O. virilis identifying changes to kinetic properties, phosphorylation state and structural stability between the enzyme from aerobic control and 20 h anoxic crayfish. Muscle AK from anoxia-exposed crayfish showed a significantly higher (by 59%) K _m for l-arginine and a lower I₅₀ value for urea than the aerobic form. Several lines of evidence indicated that AK was converted to a high phosphate form under anoxia: (a) aerobic and anoxic forms of AK showed well-separated elution peaks on DEAE ion exchange chromatography, (b) ProQ Diamond phosphoprotein staining showed a 64% higher bound phosphate content on anoxic AK compared with the aerobic form, and (c) treatment of anoxic AK with alkaline phosphatase reduced K _m l-arginine to aerobic levels whereas incubation of aerobic AK with protein kinase A catalytic subunit raised the K _m to anoxic levels. The physiological consequence of anoxia-induced AK phosphorylation may be to suppress AK activity in the phosphagen-synthesizing direction and, together with reduced cellular pH and ATP levels, promote the phosphagen-catabolizing direction under anoxic conditions. This is first time that AK has been shown to be regulated by reversible phosphorylation.     

Manipulating cytoplasmic pH under anoxia: A critical test of the role of pH in the switch from aerobic to anaerobic metabolism

Ethanol production by maize (Zea mays L.) root tips, measured by an enzymic assay of the suspending medium, was correlated with changes in the cytoplasmic pH, determined by in-vivo ³¹P nuclear magnetic resonance (NMR) spectroscopy, following the onset of anoxia. Strong evidence for the role of the cytoplasmic pH in triggering the switch to ethanol production under anoxia was obtained by: (i) varying the pH of the suspending medium between pH 4 and pH 10; and (ii) using the permeant weak base methylamine to combat the acidification of the cytoplasm induced by the anoxic conditions. Experimentally, it proved to be much easier to manipulate the cytoplasmic pH under anoxia after the pH had stabilised, rather than during the initial rapid acidification that occurred following the onset of anoxia, and in the presence of methylamine, it was possible to impose a normal aerobic cytoplasmic pH value on tissue that was metabolising anaerobically. By this means it was possible to demonstrate the reversibility of the pH effect on ethanol production under anoxia and thus to provide good evidence in support of the biochemical pH-stat model of the anoxic response. The NMR measurement of the cytoplasmic pH in the presence of methylamine was achieved by using a manganese pretreatment technique to eliminate interference between the cytoplasmic and vacuolar P_i signals, and it seems likely that the experimental approach used here will have further applications in studies of the metabolic response to anoxia.Abbreviations Caps 3-(cyclohexylamino)-1-propane sulphonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - NMR nuclear magnetic resonance - Pi inorganic phosphate We acknowledge the financial support of the Agricultural and Food Research Council and G.G.F. acknowledges the receipt of a Research Fellowship from the Royal Commission for the Exhibition of 1851.     

Centimeter-scale stream substratum heterogeneity and metabolic rates

Spatial heterogeneity of substrata in streams may influence dissolved oxygen (O₂) transport and nutrient forms. We studied the relationship between scales of substratum heterogeneity and O₂. Heterogeneous systems could have greater respiration rates as a result of increased interfacial surfaces in the biogeochemically active areas between oxic and anoxic zones. We used grids with twelve 7 × 3.5 cm cells; half the cells were filled with sand and the other half with gravel to quantify the effect of centimeter-scale heterogeneity on respiration. The sand and gravel cells were arranged within the grids to give low, medium, and high heterogeneity. Grids were incubated for 15–17 days in a prairie stream, and then whole grid respiration was analyzed in closed recirculating chambers. Depth to anoxia and substratum metabolism were calculated from O₂ microelectrode profiles measured in each cell of the grid and compared with data from natural stream transects from agricultural, urban, and prairie land use types. Shannon–Weaver (H′) diversity and “probability of change” indices were also used to compare heterogeneity of the grids to the natural stream transects. No significant differences were found among grid heterogeneity levels for respiration rate, but the anoxic interface was deeper in the gravel of higher heterogeneity grids, probably due to greater transport rates of O₂ in the coarse-grained substratum. The H′ and probability of change indices indicated that the grids had levels of heterogeneity within the range of real streams. Grid depth to anoxia and substratum metabolism rates were similar to those found in streams, though less variable. In streams, H′ and probability of change values showed a slight difference among land use types, with some urban and agricultural sites displaying very low heterogeneity. Handling editor: Robert Bailey     

High resistance of oligotrophic isoetid plants to oxic and anoxic dark exposure

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Enhanced aerobic respiration improves <Emphasis Type="Italic">in vitro</Emphasis> coconut embryo germination and culture

Summary Germination of coconut (Cocos nucifera L.) zygotic embryos was tested in liquid or solid medium. Percent germination was greater when embryos were cultured in solid medium, particularly when embryos were placed with their micropyle end facing upwards in relation to the vial orientation, independently of orientation in relation to gravity. This occurred because solid medium allowed embryos to be positioned with their micropyle end exposed to the ambient atmosphere of the vial. Germination of embryos facing upwards was suppressed when the ambient atmosphere was replaced with N₂ or when aerobic respiration inhibitors were added to the medium, whereas it was not suppressed when the atmosphere was replaced with a N₂/O₂ (50∶50, v/v) mixture, thus indicating that aerobic respiration is required for embryos to germinate. Germination in facing upwards mode also resulted in improved conversion and plantlet development. These improvements in recovery are important since they will help to avoid unintentional selection due to low in vitro germination and corresponding conversion of a population subset. These results set the basis for the improvement of current protocols for in vitro coconut embryo culture, allowing safe movement of germoplasm both in terms of phytosanitation and conservation of genetic diversity in a population of interest.     

Patterns of ubiquitylation and SUMOylation associated with exposure to anoxia in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus acquire extreme tolerance to anoxia during embryonic development. These embryos can survive environmental and cellular conditions that would likely result in death in the majority of vertebrate cells, despite experiencing a massive loss of ATP. It is highly likely that the initial response to anoxia must quickly alter cellular physiology to reprogram cell signaling and metabolic pathways to support anaerobiosis. Covalent protein modifications are a mechanism that can quickly act to effect large-scale changes in protein structure and function and have been suggested by others to play a key role in mammalian ischemia tolerance. Using Western blot analysis, we explored patterns of protein ubiquitylation and SUMOylation in embryos of A. limnaeus exposed to anoxia and anoxic preconditioning. Surprisingly, we report stage-specific protein ubiquitylation patterns that suggest different mechanisms for altering protein turnover in dormant and actively developing embryos that both survive long-term anoxia. Anoxic preconditioning does not appear to alter levels of ubiquitin conjugates in a unique manner. Global SUMOylation of proteins does not change in response to anoxia, but there are stage-specific changes in SUMOylation of specific protein bands. Contrary to other systems, global changes in protein SUMOylation may not be required to support long-term tolerance to anoxia in embryos of A. limnaeus. These data lead us to conclude that embryos of A. limnaeus respond to anoxia in a unique manner compared to other vertebrate models of anoxia tolerance and may provide novel mechanisms for engineering vertebrate tissues to survive long-term anoxia.     

Physiological energetics of a midge,Chironomus riparius Meigen (Insecta,Diptera): normoxic heat output over the whole life cycle and response of larva to hypoxia and anoxia

The rate of metabolism of laboratory reared Chironomus riparius was monitored by direct calorimetry over the entire life cycle from egg to adult stage. The metabolic response of the fourth instar larva to decreasing oxygen concentrations and anoxia was also measured. Normoxic measurements were carried out at 20°C and the hypoxic-anoxic experiments at 10°C. In larvae with body sizes ranging from 0.0028 to 0.645 mg ash-free dry mass (afdm), the rate of heat dissipation was related to body mass by a power function, with a mass exponent of 0.71±0.02 corresponding to an exponent of -0.29 for the relationship between mass-specific metabolic rate and body mass. However, the allometric equations applicable to larvae would not predict the metabolic rates of eggs, pupae and adults. Single egg batches used in the experiments consisted of 354±90 eggs, the individual egg with a mass of 0.99±0.01 g (mean±SD). The mass-specific rate of heat dissipation of the egg (13.7±1.8 W mg^-1 afdm) was considerably lower than that of the first and second instar larvae (44–53 W mg^-1) but equal to that of fourth instar larvae (13.1±3.9 W mg^-1). Heat dissipation by a pupa shortly before adult emergence was high (14.8±1.8 W mg^-1), probably due to high metabolism during metamorphosis. Emergence of the adult in the calorimeter was indicated by a short but intense burst of heat. The newly emerged imago had a ca. 20–35% higher metabolic rate than the pupa. In response to reduced O₂ partial pressure the fourth instar larva of C. riparius displayed metabolic regulation. In continuously declining oxygen partial pressure, the fourth instar larva maintained its aerobic energy metabolism (4.2 W mg^-1) with only a small decrease down to 0.8 kPa, corresponding to an oxygen concentration of 0.42 mg O₂l^-1 H₂O. Below this critical oxygen concentration (P_c), the rate of heat dissipation decreased rapidly down to the anoxic level which was only 14–17% of the normoxic level. The high relative reduction of metabolic rate under anoxia gives a wrong impression of short-term tolerance of C. riparius to anoxia. The absolute energetic costs of C. riparius associated with anaerobic energy metabolism (0.64±0.11 W mg^-1) are almost 6 times higher than those of more anoxia tolerant invertebrates such as sphaeriid bivalves.     

The effects of hypoxia and temperature on metabolic aspects of embryonic development in the annual killifish Austrofundulus limnaeus

Embryos of Austrofundulus limnaeus are exceptional in their ability to tolerate prolonged bouts of complete anoxia. Hypoxia and anoxia are a normal part of their developmental environment. Here, we exposed embryos to a range of PO₂ levels at two different temperatures (25 and 30 °C) to study the combined effects of reduced oxygen and increased temperature on developmental rate, heart rate, and metabolic enzyme capacity. Hypoxia decreased overall developmental rate and caused a stage-specific decline in heart rate. However, the rate of early development prior to the onset of organogenesis is insensitive to PO₂. Increased incubation temperature caused an increase in the developmental rate at high PO₂s, but hindered developmental progression under severe hypoxia. Embryonic DNA content in pre-hatching embryos was positively correlated with PO₂. Citrate synthase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase capacity were all reduced in embryos developing under hypoxic conditions. Embryos of A. limnaeus are able to develop normally across a wide range of PO₂s and contrary to most other vertebrates severe hypoxia is not a teratogen. Embryos of A. limnaeus do not respond to hypoxia through an increase in the capacity for enzymatic activity of the metabolic enzymes lactate dehydrogenase, citrate synthase, or phosphoenolpyruvate carboxykinase. Instead they appear to adjust whole-embryo metabolic capacity to match oxygen availability. However, decreased DNA content in hypoxia-reared embryos suggests that cellular enzymatic capacity may remain unchanged in response to hypoxia, and the reduced capacity may rather indicate reduced cell number in hypoxic embryos.     

Metabolic suppression in anoxic frog muscle

Microcalorimetry is the only direct method for measuring moment-to-moment changes in whole-cell metabolism (as heat output) during anoxia. We have adapted this methodology, in conjunction with standard muscle isolation techniques, to monitor metabolic transitions in isolated frog (Rana temporaria) sartorius muscle during anoxia and recovery (reoxygenation). Anoxia (sustained 1 h, following 2 h progressive hypoxia) suppressed muscle heat output to 20% of the stable normoxic level. This effect was fully reversible upon reoxygenation. Metabolite profiles were consistent with other anoxia-tolerant vertebrates – most notably, adenosine triphosphate (ATP) content during anoxia and reoxygenation remained unchanged from normoxia (pre-anoxic control). In addition, the concentration of K⁺ ions ([K⁺]) in interstitial dialysates remained stable (2–3 mM) throughout anoxia and recovery. Interstitial [lactate⁻] increased slightly, in accord with anaerobiosis supporting suppressed metabolic rates during anoxia. The degree of anoxic suppression of metabolism observed is similar to other vertebrate models of anoxia tolerance. Furthermore, stable ATP concentrations and interstitial [K⁺] in the isolated tissue suggests that intrinsic mechanisms suppress metabolism in a manner that coordinates ATP supply and demand and avoids the severe ion imbalances that are characteristic of hypoxia-sensitive systems. Accepted: 15 January 1998     

Developmental and metabolic changes induced by anoxia in diapausing and non-diapausing flesh fly pupae

Summary Anaerobic metabolism was compared in nondiapausing (ND) and diapausing (D) pupae of the flesh fly,Sarcophage crassipalpis using in vivo¹³C NMR spectroscopy. Anoxia-induced changes in the development of ND and D pupae were correlated with oxidative metabolism and mitochondrial integrity. ND pupae tolerated 1 day of anoxia without any obvious developmental effect, while D pupae tolerated up to 6 days of anoxia. Longer exposure to anoxia (3 days in ND pupae and up to 14 days in D pupae) allowed development to the pharate adult stage but precluded eclosion. Four-day anoxia applied to ND pupae during 4–6 days post-pupariation arrested development in a stage indistinguishable from diapause. This morphological stasis was accompanied by 80% suppression of oxidative metabolism and a 100% increase in glycerol concentration. However, unlike a true diapause, this arrest could not be terminated with 20-hydroxyecdysone or hexane. Four-day anoxia treatment applied to D pupae stimulated development and raised their oxygen consumption. The anoxia-induced changes in oxidative metabolism were not accompanied by mitochondrial changes. Exposure to 95%PO₂ atmosphere had no apparent developmental or metabolic effects on ND or D pupae. Major metabolites (lipids, trehalose, glycogen, glycerol, glutamine, and alanine) were detected in the ND and D pupae but their rates of turnover differed. Anoxia induced synthesis of glycerol and alanine in both D and ND pupae. Injected labeled glucose was incorporated primarily into trehalose and glycogen by both D and ND pupae. The rate of incorporation in ND pupae was approximately twice that observed in D pupae. Anoxia resulted in glycerol and alanine synthesis in both groups of pupae, but more glycerol was labeled in ND pupae and more alanine in D pupae. Glycogen and trehalose were depleted in the D pupae under anoxia. Cold acclimation had no effect on the steady-state or rate of synthesis of metabolites.     

Effects of sediment anoxia and light on turion germination and early growth of Potamogeton crispus

Potamogeton crispus is a cosmopolitan aquatic species and is widely used as a pioneer species for vegetation restoration of eutrophic lakes. However, many restoration projects applying P. crispus turions have not been successful. Earlier studies focused on effects of light and temperature on turion germination. The purpose of this study was to determine whether sediment anoxia and light interactively affected the turion germination and early growth of P. crispus. Anoxic conditions in the experiment were produced by adding sucrose to the sediment. The germination rate of the turions was 68–73% lower in the highly anoxic condition treatment than in the control. Medium light intensity (10% of natural light at the water surface) was more favorable for germination under slightly anoxic conditions than either low or high light intensity. The growth of newly-formed sprouts was also significantly inhibited by sediment anoxia. Photosynthesis and shoot biomass were reduced under sediment anoxia, whereas total chlorophyll content, root biomass, and soluble protein content were highest in the low anoxic condition treatment. Medium light improved net photosynthesis and biomass production of the sprouts. We conclude that turion germination and sprout growth can be significantly inhibited by sediment anoxia. Medium light intensity may alleviate this inhibition by anoxia, but light has little effect when sediment anoxia is severe. For the purposes of vegetation restoration, more attention should be paid to the role of sediment anoxia, and it is necessary to improve sediment and light conditions for turion germination and early growth of P. crispus in eutrophic lakes. These results will contribute to a more complete understanding of turion germination dynamics of P. crispus and will be useful for future restoration programs. Handling editor: S. M. Thomaz