Jonathan C. Wright

CRYPTOBIOSIS IN TARDIGRADA

CONTENTS

Cryptobiosis 300 Years on from van Leuwenhoek: What Have We Learned about Tardigrades?

Biochemical and ultrastructural advances in the latter part of the 20th C. have revolutionized our understanding of cryptobiosis since Anton van Leeuwenhoek gave the first formal description of the phenomenon at a Royal Society lecture in 1702. Keilin coined the term cryptobiosis in 1959 to describe the entry into a reversible ametabolic state and recognized that such ‘latent life’ could encompass processes induced by dehydration, cooling, and perhaps osmotic stress and anoxia. True cryptobiosis, as now understood, depends on the loss of a liquid water phase and can be induced by desiccation or freezing. Loss of ‘bulk’ or liquid water may result directly from evaporation, or arise through vitrification promoted by the formation of a carbohydrate matrix. The carbohydrates in question appear to be ubiquitous in cryptobiotes and serve multiple additional roles: as compatible intracellular osmolytes during desiccation or freeze-dehydration; as stabilizers of protein quaternary structure and lipid bilayer integrity with declining free water activity; and as supercoolants. Plants tend to rely on oligosaccharides such as stachyose and raffinose, while yeasts, spores, and metazoans depend primarily on disaccharides, particularly sucrose and trehalose, and on glycerol. As in many other anhydrobiotes, a metabolic preparatory stage in which these carbohydrates are synthesized from glycogen reserves appears essential for anhydrobiosis in tardigrades, and thus limits physiological tolerance of desiccation rate. Adaptive processes such as tun-formation in tardigrades and bdelloids, coiling in nematodes, and gradient-dependent changes in integumental permeability, retard water losses during preliminary desiccation and exert important influence on survival in xeric extremes. In tardigrades, cryobiosis, or cold-induced cryptobiosis, differs from anhydrobiosis in several important details. Tun formation is not essential for survival, and tolerance of cooling rate depends on the ability to inhibit intracellular freezing. Unlike many cold-tolerant arthropods, tardigrades are freeze-tolerant. Extracellular freezing is promoted by one or more ice-nucleating proteins in Adorybiotus coronifer, and occurs at high temperatures close to 0 °C. Tolerance of variable cooling rates to sub-freezing temperatures in this species does not seem to depend on trehalose synthesis, although a role of other possible intracellular cryoprotectants is likely. It is presently unclear whether cryobiotic tardigrades undergo cytoplasmic vitrification, or whether freeze-dehydration and colligative lowering of cytoplasmic water activity renders the remaining water unfreezable. The profound tolerance of environmental extremes displayed by cryptobiotic organisms apparently depends on the loss of a liquid water phase with accompanying metabolic depression as elegantly described by Cleggs vicinal network model. Thus protected from temperature- and solute-dependent effects on reaction kinetics, and (in part) from destructive free-radical oxidation, cryptobiotes can retain viability in a near-inert state for decades. Disruption of multi-subunit enzyme reactions accompanying loss of the vicinal water fraction may control the decline in metabolism, but also eliminates important free-radical scavenging pathways. Glycerol partly offsets this, acting as an antioxidant, but progressive free-radical oxidation in cryptobiotes may set the upper limit to longevity under aerobic conditions. While we can make many inferences of the physiology of cryptobiosis in tardigrades based on information gathered from other cryptobiotic organisms, specific studies on tardigrades are few, and encompass only a small number of species. This should prove a fruitful field for future research.

Embryo Tolerance and Maternal Control of the Marsupial Environment in Armadillidium vulgare (Isopoda: Oniscidea)

Marsupial development in terrestrial isopods subjects embryos to potential physiological stresses, including desiccation, osmotic variation, and high ammonia concentrations. In this study, we investigated tolerance of osmotic extremes, total ammonia, and pH in developmental stages of Armadillidium vulgare cultured in vitro. Marsupial stages were classified as stage 1 (chorionated eggs), stage 2 (having shed the chorion), and stage 3 (mancas). All stages showed wide but differing tolerance ranges. Stage 1 eggs possess the greatest ammonia tolerance, with high 7‐d survival in 150 mM total ammonia, and a wide pH tolerance range. Mancas show the widest osmotic tolerance (100–1,400 mosm kg−1) and display proficient hemolymph osmoregulation over this range. Stage 2 eggs reveal the narrowest tolerance ranges for all three parameters but still qualify as eurytopic. Silver staining revealed two distinct ion‐transporting tissues in the developmental stages: a median band on the vitelline membrane of stage 1 and stage 2 eggs, corresponding in location to the embryonic dorsal organ, and the posterior three pairs of pleopodal endopodites in mancas. Gravid females do not downregulate ammonia but show efficient regulation of marsupial fluid pH and downregulation of osmolality during dehydration, both of which will provide additional protection to the marsupial young.

Postprandial energy expenditure in whole-food and processed-food meals: implications for daily energy expenditure

Background Empirical evidence has shown that rising obesity rates closely parallel the increased consumption of processed foods (PF) consumption in USA. Differences in postprandial thermogenic responses to a whole-food (WF) meal vs. a PF meal may be a key factor in explaining obesity trends, but currently there is limited research exploring this potential link. Objective The goal was to determine if a particular PF meal has a greater thermodynamic efficiency than a comparable WF meal, thereby conferring a greater net-energy intake. Design Subjective satiation scores and postprandial energy expenditure were measured for 5–6 h after isoenergetic meals were ingested. The meals were either ‘whole’ or ‘processed’ cheese sandwiches; multi-grain bread and cheddar cheese were deemed whole, while white bread and processed cheese product were considered processed. Meals were comparable in terms of protein (15–20%), carbohydrate (40–50%), and fat (33–39%) composition. Subjects were healthy women (n=12) and men (n=5) studied in a crossover design. Results There were no significant differences in satiety ratings after the two meals. Average energy expenditure for the WF meal (137±14.1 kcal, 19.9% of meal energy) was significantly larger than for the PF meal (73.1±10.2 kcal, 10.7% of meal energy). Conclusion Ingestion of the particular PF meal tested in this study decreases postprandial energy expenditure by nearly 50% compared with the isoenergetic WF meal. This reduction in daily energy expenditure has potential implications for diets comprised heavily of PFs and their associations with obesity.

Atmospheric Water Absorption and the Water Budget of Terrestrial Isopods (Crustacea, Isopoda, Oniscidea)

Studies of terrestrial isopods (Crustacea, Isopoda, Oniscidea) have revealed a capacity for active water vapor absorption (WVA) in the taxonomic sections Crinocheta and Diplocheta but not in Synocheta. Uptake thresholds in Crinocheta are modest by comparison with other vapor absorbers, but standardized uptake fluxes are among the highest recorded and are probably an adaptive requirement to counter the high transpiratory losses. Comparative data for uptake fluxes, thresholds, and transpiratory losses allows the compilation of water budgets in hypothetical temperature and humidity regimes. Given a 12-h light-dark cycle, with saturated ambient activities for diurnal WVA, all species could recover water losses incurred during nocturnal foraging in an ambient water activity of 0.75, and xeric species could forage in activities below 0.30. Xeric trends based on these models agree closely with predictions from ecotypic surveys. In the littoral Ligia oceanica (Diplocheta) haemolymph hyperosmosis and periodic submergence provide additional means of water balance regulation. It is proposed that WVA in Ligia provides an essentially solute-free water source to counteract salt-loading in the splash-zone. The absence of WVA in synochetes, together with their cryptozoic habits, reflects an alternative terrestrial strategy to those of other oniscideans.

Haemolymph osmoregulation and the fate of sodium and chloride during dehydration in terrestrial isopods

Osmoregulation of the haemolymph during dehydration was investigated in a selection of temperate oniscidean isopods. Inulin tracer studies show that the haemolymph contributes approximately 69% of water losses in Porcellio scaber, significantly more than predicted from the volume of this compartment (42% of total water). Haemolymph osmolality increases linearly as a function of haemolymph dehydration but at a significantly lower rate than predicted from the change in haemolymph fluid volume. Similar results for Oniscus asellus show that both species display efficient osmoregulation until lethal dehydration. Osmoregulation is associated with significant hyporegulation of haemolymph sodium and chloride. These findings indicate that: (1) cell water is conserved at the expense of the haemolymph; and (2) haemolymph dehydration is associated with the removal of Na(+) and Cl(-) contributing to net osmoregulation. During dehydration, accumulations of both Na(+) and Cl(-) are seen in the hindgut, with significant accumulations of electrolytes also seen in the luminal fluid of the hepatopancreas. Low fluid volumes in the foregut and hindgut suggest macromolecular association as the most plausible mechanism of ion sequestration. Evidence refutes ion excretion and haemocyte sequestration as osmoregulatory mechanisms. Sequestration of Na(+) as urate salts, as shown for Periplaneta and generally assumed for other insects, is insignificant in isopods.

Kinematic Evidence for Superfast Locomotory Muscle in Two Species of Teneriffiid Mites

SUMMARY Locomotory muscles typically operate over a narrow range of contraction frequencies, characterized by the predominant fiber types and functional roles. The highest documented frequencies in the synchronous sound-producing muscles of insects (550 Hz) and toadfish (200 Hz) far exceed the contraction frequencies observed in weight-bearing locomotory muscles, which have maximum documented frequencies below 15–30 Hz. Laws of scaling, however, predict that smaller arthropods may employ stride frequencies exceeding this range. In this study we measured running speed and stride frequency in two undescribed species of teneriffiid mites from the coastal sage scrub of southern California. Relative speeds of both species [129–133 body lengths (BL)s–1] are among the fastest documented for any animal. Measured stride frequencies for both species far exceed those documented for any weight-bearing locomotory muscle, with measured values for one species ranging from 93 Hz at 25°C to 111 Hz at 45°C. Stride frequencies either closely approximate or, for one species, exceed predicted values based on an interspecific scaling of frequency and animal mass. Consequently, while the ultra-high frequencies of these muscles must depend on appropriately scaled kinetics of the calcium transient and contraction–relaxation cycle, these do not appear to limit the operating frequencies during running. The predicted low muscle forces operating at these very high frequencies evidently suffice for locomotion, probably because of the larger relative muscle force generated by smaller animals.

Energy-Dependent Water Vapor Absorption (WVA) in the Pleoventral Cavity of Terrestrial Isopods (Crustacea, Isopoda, Oniscidea): Evidence for Pressure Cycling as a Supplement to the Colligative Uptake Mechanism

Terrestrial isopods (Crustacea, Isopoda, Oniscidea) are capable of active water vapor absorption from subhemolymph water activities. The minimum activity for absorption constitutes the uptake threshold (T), and animals can balance passive losses above the critical equilibrium activity (CEA). Values for Porcellio scaber are 0.889 and 0.913, respectively. Two major mechanisms can explain the uptake process. Maximum attainable uptake fluxes increase linearly with ambient water activity (Aaw) although animals are capable of depressing uptake fluxes below, this curve. Such kinetics are compatible with a steady-state secretion/resorption of hyperosmotic fluid in the pleoventral cavity (PV) and ventilation of ambient air by the pleopodal exopods. At near-threshold activities animals may also reveal sharply augmented uptake fluxes. Comparisons with available data 0on fluid concentrations, and direct observations of pleopodal ventilation, indicate the elevation of Aaw within the PV by pressure cycling. This is confirmed by monitoring pressure fluctuations, coincident with pleopodal ventilations, in an enclosed chamber. Blocking experiments support endogenous secretion/resorption of pleon fluid. Fecal activities are approximately 0.990, discounting a rectal resorption mechanism based on transepithelial activity gradients.

Ultrastructural and histochemical investigations of peripatus integument

The integument of Euperipatoides leukarti (Onychophora) has been studied by SEM and TEM-histochemical techniques. The thin (2-3 mu) cuticle is essentially arthropodan in design, comprising a thin, lamellate outer epicuticle, a homogeneous inner epicuticle, and an extensive fibrous procuticle. Five tanned lipoprotein lamellae constitute the outer epicuticle, the innermost showing a cross-striated pattern and the outermost also containing carbohydrates. The inner epicuticle contains untanned lipoproteins. The chitin-protein composite procuticle reveals a fibrous ultrastructure but no trace of helicoids. Distally it may be tanned, producing a sclerotin layer (exocuticle), pronounced in the sensory setae, claws and mandibles. Penetrating the epicuticle and extending into the procuticle are numerous, minute pore canals through which mobilised procuticle is resorbed prior to ecdysis. Beneath the procuticle is a monolayered epidermis traversed by tonofibrils and containing dense pigment granules. The membrane junctions are strongly convoluted and include an apical zonula adherens, septate desmosomes and a proximal lymph space. The epidermis is underlain by a thick (5-25mu) basement membrane of helicoidally organised collagen fibres. Functional and phylogetic implications of the integumental structure are discussed. Arthropod affinities are clear and the Onychophora should be included in that phylum if it is to be retained in modern taxonomic nomenclature.

Increases in tissue amino acid levels in response to ammonia stress in the terrestrial isopod Porcellio scaber Latr.

Terrestrial isopods excrete waste nitrogen by intermittent volatilization of ammonia and must therefore accumulate amino groups in a non-toxic form between excretory bouts. High-performance liquid chromatography (HPLC) was used to analyze concentrations of 23 amino acids in the oniscidean Porcellio scaber following a 7-day exposure to elevated PNH3 (“ammonia stressing”). Analyses were performed on four separate tissues: hindgut, hepatopancreas, pleopodal endopods, and the body wall. Concentrations of all free amino acids except histidine and cysteine increased in ammonia-stressed animals. The greatest whole-animal concentrations (30–40 μmol·g fw−1) were seen in glutamine and glycine, which also showed the largest increases (fourfold). Most glutamine and glycine is accumulated in the body wall and hepatopancreas with concentrations in the hepatopancreas reaching 400–500 μmol·g fw−1. Ammonia-stressed animals showed smaller but substantial accumulations of alanine, arginine, proline, and glutamate. Arginine, by virtue of its 3N R-group, constitutes the major nitrogen-storage compound, together with glutamine. Calculations based on these and our previous data indicate that the amino acids constitute the primary form in which nitrogen is accumulated under ammonia-stressing conditions.