Surviving the cold: molecular analyses of insect cryoprotective dehydration in the Arctic springtail Megaphorura arctica (Tullberg)

<p>Abstract</p> <p>Background</p> <p>Insects provide tractable models for enhancing our understanding of the physiological and cellular processes that enable survival at extreme low temperatures. They possess three main strategies to survive the cold: freeze tolerance, freeze avoidance or cryoprotective dehydration, of which the latter method is exploited by our model species, the Arctic springtail <it>Megaphorura arctica</it>, formerly <it>Onychiurus arcticus </it>(Tullberg 1876). The physiological mechanisms underlying cryoprotective dehydration have been well characterised in <it>M. arctica </it>and to date this process has been described in only a few other species: the Antarctic nematode <it>Panagrolaimus davidi</it>, an enchytraied worm, the larvae of the Antarctic midge <it>Belgica antarctica </it>and the cocoons of the earthworm <it>Dendrobaena octaedra</it>. There are no in-depth molecular studies on the underlying cold survival mechanisms in any species.</p> <p>Results</p> <p>A cDNA microarray was generated using 6,912 <it>M. arctica </it>clones printed in duplicate. Analysis of clones up-regulated during dehydration procedures (using both cold- and salt-induced dehydration) has identified a number of significant cellular processes, namely the production and mobilisation of trehalose, protection of cellular systems via small heat shock proteins and tissue/cellular remodelling during the dehydration process. Energy production, initiation of protein translation and cell division, plus potential tissue repair processes dominate genes identified during recovery. Heat map analysis identified a duplication of the trehalose-6-phosphate synthase (TPS) gene in <it>M. arctica </it>and also 53 clones co-regulated with TPS, including a number of membrane associated and cell signalling proteins. Q-PCR on selected candidate genes has also contributed to our understanding with glutathione-S-transferase identified as the major antioxdidant enzyme protecting the cells during these stressful procedures, and a number of protein kinase signalling molecules involved in recovery.</p> <p>Conclusion</p> <p>Microarray analysis has proved to be a powerful technique for understanding the processes and genes involved in cryoprotective dehydration, beyond the few candidate genes identified in the current literature. Dehydration is associated with the mobilisation of trehalose, cell protection and tissue remodelling. Energy production, leading to protein production, and cell division characterise the recovery process. Novel membrane proteins, along with aquaporins and desaturases, have been identified as promising candidates for future functional analyses to better understand membrane remodelling during cellular dehydration.</p

Flavopiridol Pharmacogenetics: Clinical and Functional Evidence for the Role of SLCO1B1/OATP1B1 in Flavopiridol Disposition

Flavopiridol is a cyclin-dependent kinase inhibitor in phase II clinical development for treatment of various forms of cancer. When administered with a pharmacokinetically (PK)-directed dosing schedule, flavopiridol exhibited striking activity in patients with refractory chronic lymphocytic leukemia. This study aimed to evaluate pharmacogenetic factors associated with inter-individual variability in pharmacokinetics and outcomes associated with flavopiridol therapy.Thirty-five patients who received single-agent flavopiridol via the PK-directed schedule were genotyped for 189 polymorphisms in genes encoding 56 drug metabolizing enzymes and transporters. Genotypes were evaluated in univariate and multivariate analyses as covariates in a population PK model. Transport of flavopiridol and its glucuronide metabolite was evaluated in uptake assays in HEK-293 and MDCK-II cells transiently transfected with SLCO1B1. Polymorphisms in ABCC2, ABCG2, UGT1A1, UGT1A9, and SLCO1B1 were found to significantly correlate with flavopiridol PK in univariate analysis. Transport assay results indicated both flavopiridol and flavopiridol-glucuronide are substrates of the SLCO1B1/OATP1B1 transporter. Covariates incorporated into the final population PK model included bilirubin, SLCO1B1 rs11045819 and ABCC2 rs8187710. Associations were also observed between genotype and response. To validate these findings, a second set of data with 51 patients was evaluated, and overall trends for associations between PK and PGx were found to be consistent.Polymorphisms in transport genes were found to be associated with flavopiridol disposition and outcomes. Observed clinical associations with SLCO1B1 were functionally validated indicating for the first time its relevance as a transporter of flavopiridol and its glucuronide metabolite. A second 51-patient dataset indicated similar trends between genotype in the SLCO1B1 and other candidate genes, thus providing support for these findings. Further study in larger patient populations will be necessary to fully characterize and validate the clinical impact of polymorphisms in SLCO1B1 and other transporter and metabolizing enzyme genes on outcomes from flavopiridol therapy

Surviving extreme polar winters by desiccation: clues from Arctic springtail (Onychiurus arcticus) EST libraries

Background Ice, snow and temperatures of -14°C are conditions which most animals would find difficult, if not impossible, to survive in. However this exactly describes the Arctic winter, and the Arctic springtail Onychiurus arcticus regularly survives these extreme conditions and re-emerges in the spring. It is able to do this by reducing the amount of water in its body to almost zero: a process that is called "protective dehydration". The aim of this project was to generate clones and sequence data in the form of ESTs to provide a platform for the future molecular characterisation of the processes involved in protective dehydration. Results Five normalised libraries were produced from both desiccating and rehydrating populations of O. arcticus from stages that had previously been defined as potentially informative for molecular analyses. A total of 16,379 EST clones were generated and analysed using Blast and GO annotation. 40% of the clones produced significant matches against the Swissprot and trembl databases and these were further analysed using GO annotation. Extraction and analysis of GO annotations proved an extremely effective method for identifying generic processes associated with biochemical pathways, proving more efficient than solely analysing Blast data output. A number of genes were identified, which have previously been shown to be involved in water transport and desiccation such as members of the aquaporin family. Identification of these clones in specific libraries associated with desiccation validates the computational analysis by library rather than producing a global overview of all libraries combined. Conclusion This paper describes for the first time EST data from the arctic springtail (O. arcticus). This significantly enhances the number of Collembolan ESTs in the public databases, providing useful comparative data within this phylum. The use of GO annotation for analysis has facilitated the identification of a wide variety of ESTs associated with a number of different biochemical pathways involved in the dehydration and recovery process in O. arcticus

Cold hardening processes in the Antarctic springtail, Cryptopygus antarcticus: clues from a microarray.

The physiology of the Antarctic microarthropod, Cryptopygus antarcticus, has been well studied, particularly with regard to its ability to withstand low winter temperatures. However, the molecular mechanisms underlying this phenomenon are still poorly understood. 1180 sequences (Expressed Sequence Tags or ESTs) were generated and analysed, from populations of C. antarcticus. This represents the first publicly available sequence data for this species. A sub-set (672 clones) were used to generate a small microarray to examine the differences in gene expression between summer acclimated cold tolerant and non-cold tolerant springtails. Although 60% of the clones showed no sequence similarity to annotated genes in the datasets, of those where putative function could be inferred via database homology, there was a clear pattern of up-regulation of structural proteins being associated with the cold tolerant group. These structural proteins mainly comprised cuticle proteins and provide support for the recent theory that summer SCP variation within Collembola species could be a consequence of moulting, with moulting population having lowered SCP

Spatial and temporal variability of ground surface temperature and active layer thickness at the margin of maritime Antarctica, Signy Island

A CALM grid with a data logger system to monitor the active layer thermal regime was established on Signy Island (60°43′S, 45°38′Wat 80 m a.s.l.) in December 2005. The active layer at each of the 36 nodes of the grid was monitored measuring the ground temperature at least at 4 different depths between 0.02 and 0.4 m at the end of the summer season. In addition, within the grid, we selected four sites closely spaced (in a ray of 25 m) three of which with the same topographical characteristics (north facing aspect) but different vegetation coverage (one bare ground, BG1 and two sites with different vegetation: Andreaea sp. and Sanionia uncinata) and the fourth (BG2) it is as BG1 a bare ground but with south facing aspect. In particular, 4 thermistors were located at depths of 0.02, 0.3, 0.6, and 0.9 m at BG2 and at the Andreaea sp site, 9 thermistors at 0.02, 0.3, 0.6, 1, 1.2, 1.4, 1.6, 2, and 2.5 mat BG1 and at 0.02 and 0.6 mof depth at Sanionia site. Generally, with the same aspect, a thick vegetation cover (as in Sanionia site) provides a greater insulative effect than a thinner vegetation cover (as in Andreaea site) or bare ground (BG1) because vegetation both shades and insulates the ground resulting in a reduction in summer heat flux. Ground Surface Temperature (GST) was colder and more buffered in spring and summer under the vegetated ground than in BG1, although the coldest GST and lowest Thawing Degree Days (TDD) were recorded at BG2 and related to its southern aspect. Our data confirm that air temperature is the main driver of GST, as already reported both in the Arctic and Antarctic. We also found that the effect of air temperature changes seasonally, being drastically reduced in winter and, to a lesser extent, in fall and spring, when there is generally thin snow cover (b30 cm). During the summer, when snow cover is usually absent, the air temperature is the dominant driver, although incoming radiation also had an effect on the northern exposed bare ground and to a lesser extent on the vegetated and southerly exposed bare ground. The active layer ranges between 81 and 185 cm on the 4 continuously monitored sites and, considering the sites with the same aspect, it is thicker under bare ground (between 10% up to more than 100%) than under vegetated ground, confirming previous observations in the Arctic and Antarctic. However at our sites, climate forcing has no effect on the active layer thickness, enhancing the role of soil properties including the periods of high moisture content and lateral flow of water. The lack of a statistically significant regressions between GST and active layer thickness could be due to the limited study period (four years) and/or to the variationwith timeof changes in soil characteristics such as soilmoisture, and the possible occurrence of non-conductive heat transfer processes including the lateral flow of water. Further data are required to understand the role of moisture and possible ground water circulation within the active layer to explain the unexpected strong dichotomy between the GST regime and active layer thickness

Assemblage level variation in springtail lower lethal temperature: the role of invasive species on sub-Antarctic Marion Island

It is widely held both in the physiological literature, and more generally, that the average characteristics of species within an assemblage differ among sites. Such generalizations should be based on investigations of whole assemblages at sites, but this is rarely done. Here, such a study is undertaken for virtually the full assemblage of springtails found at sub-Antarctic Marion Island, by investigating supercooling points (SCPs) of 12 of the 16 species that occur there. Assemblage level variation tends to be less than that documented for assemblages across northern hemisphere sites but similar to that found at some Antarctic locations. Across this set of species, the mean SCPs of the indigenous species (mean +/- SE = -17.2 +/- 0.4 degrees C) do not differ significantly from that of the invasive species (-16.3 +/- 0.7 degrees C). Overall, the introduction of several species to the island does not appear to have led to functional homogenization (for this trait). By combining the assemblage-level SCP data with information on the abundances of the species in each of four major habitats, it is also shown that severe but uncommon low temperature events could substantially alter species relative abundances. By resetting assemblage trajectories, such events could play an important role in the terrestrial system at the island

Gene expression associated with changes in cold tolerance levels of the Antarctic springtail, Cryptopygus antarcticus

The ability of the Antarctic microarthropod Cryptopygus antarcticus (Collembola, Isotomidae) to survive low temperatures has been well studied at the physiological level, with recent investigations indicating the importance of the moulting process in conferring this ability. This study investigated gene expression in groups of C. antarcticus that have distinct differences in their ability to survive low temperatures. A microarray containing c. 5400 C. antarcticus expressed sequence tags was used to investigate gene expression differences between groups of animals with different supercooling points (SCP), and to low temperatures close to their SCP. By demonstrating the involvement of moult-related genes in the differential survival of two groups of C. antarcticus with distinct SCP profiles, the results of this investigation add support to the suggestion that moulting plays a role in conferring cold tolerance in C. antarcticus

High-temperature tolerance in anhydrobiotic tardigrades is limited by glass transition

Survival in microhabitats that experience extreme fluctuations in water availability and temperature requires special adaptations. To withstand such environmental conditions, tardigrades, as well as some nematodes and rotifers, enter a completely desiccated state known as anhydrobiosis. We examined the effects of high temperatures on fully desiccated (anhydrobiotic) tardigrades. Nine species from the classes Heterotardigrada and Eutardigrada were exposed to temperatures of up to 110 degrees C for 1 h. Exposure to temperatures of up to 80 degrees C resulted in a moderate decrease in survival. Exposure to temperatures above this resulted in a sharp decrease in survival, with no animals of the families Macrobiotidae and Echiniscidae surviving 100 degrees C. However, Milnesium tardigradum (Milnesidae) showed survival of >90% after exposure to 100 degrees C; temperatures above this resulted in a steep decrease in survival. Vitrification is assumed to play a major role in the survival of anhydrobiotic organisms during exposure to extreme temperatures, and consequently, the glass-transition temperature (T-g) is critical to high-temperature tolerance. In this study, we provide the first evidence of the presence of a glass transition during heating in an anhydrobiotic tardigrade through the use of differential scanning calorimetry

Freeze tolerance, supercooling points and ice formation: comparative studies on the subzero temperature survival of limno-terrestrial tardigrades

Many limno-terrestrial tardigrades live in unstable habitats where they experience extreme environmental conditions such as drought, heat and subzero temperatures. Although their stress tolerance is often related only to the anhydrobiotic state, tardigrades can also be exposed to great daily temperature fluctuations without dehydration. Survival of subzero temperatures in an active state requires either the ability to tolerate the freezing of body water or mechanisms to decrease the freezing point. Considering freeze tolerance in tardigrades as a general feature, we studied the survival rate of nine tardigrade species originating from polar, temperate and tropical regions by cooling them at rates of 9, 7, 5, 3 and 1 degrees Ch(-1) down to -30 degrees C then returning them to room temperature at 10 degrees Ch(-1). The resulting moderate survival after fast and slow cooling rates and low survival after intermediate cooling rates may indicate the influence of a physical effect during fast cooling and the possibility that they are able to synthesize cryoprotectants during slow cooling. Differential scanning calorimetry of starved, fed and cold acclimatized individuals showed no intraspecific significant differences in supercooling points and ice formation. Although this might suggest that metabolic and biochemical preparation are non-essential prior to subzero temperature exposure, the increased survival rate with slower cooling rates gives evidence that tardigrades still use some kind of mechanism to protect their cellular structure from freezing injury without influencing the freezing temperature. These results expand our current understanding of freeze tolerance in tardigrades and will lead to a better understanding of their ability to survive subzero temperature conditions