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

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

Identification of a metallothionein gene in honey bee Apis mellifera and its expression profile in response to Cd, Cu and Pb exposure.

Metallothioneins are ubiquitous proteins important in metal homeostasis and detoxification. However, they have not previously been identified in honey bees or other Hymenoptera, where metallothioneins could be of ecophysiological and ecotoxicological significance. Better understanding of the molecular responses to stress induced by toxic metals could contribute to honey bee conservation. In addition, honey bee metallothionein could represent a biomarker for monitoring environmental quality. Here we identify and characterize a metallothionein gene in Apis mellifera (AmMT). AmMT is 1,680 bp long and encodes a 48 amino acids protein with 15 cysteines and no aromatic residues. A metal response element upstream of the start codon, coupled with numerous cis-regulatory elements indicate the functional context of AmMT. Molecular modelling predicts several transition metal binding sites, and comparative phylogenetic analysis revealed five putative metallothionein proteins in three other hymenoptera species. AmMT was characterized by cloning the full-length coding sequence of the putative metallothionein. Recombinant AmMT was found to increase metal tolerance upon overexpression in Escherichia coli supplemented with Cd, Cu or Pb. Finally, in laboratory tests on honey bees, gene expression profiles showed a dose-dependant relationship between Cd, Cu and Pb concentrations present in food and AmMT expression, while field experiments showed induction of AmMT in bees from an industrial site compared to those from an urban area. These studies suggest that AmMT has metal binding properties in agreement with a possible role in metal homeostasis. Further functional and structural characterization of metallothionein in honey bees and other Hymenoptera are necessary

Oxidative stress and the activity of antioxidative defense enzymes in overwintering honey bees

Over the past decades, the number of managed honey bee Apis mellifera L. (Hymenoptera: Apidae) colonies have been decreasing. The majority of losses occur during winter, suggesting that overwintering honey bees are more susceptible to adverse factors. We focused on the oxidative status of overwintering honey bees, particularly at the beginning (November) and end (March) of the wintering period. Colonies from three locations with different anthropogenic influences were selected: Belgrade, an urban zone, Zajača, an industrial zone, and Susek, a rural area. We measured levels of malondialdehyde (MDA), as a marker of lipid peroxidation, as well as the expression and activity of select antioxidative enzymes: superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST). Our results show that enzyme activity and gene expression of antioxidative enzymes are influenced by both sample location and the time of sampling. The majority of analyzed genes had significantly reduced expression, at the end of the overwintering period when higher activities of antioxidative enzymes were also recorded. Among the analyzed parameters, SOD activity and gene expression of microsomal GST isoforms were more affected by local environmental conditions, suggesting the complex role of these enzymes in antioxidative defense and detoxification. The higher MDA levels observed at the end of overwintering for all three locations likely reflects accumulated oxidative damage which could be associated with the aging process, brood rearing and/or the onset flying activity

Maximum parsimony tree of 12 aquaporin genes and the three clones (sb 005 09I19, CL138 and sb 006 05H07) (arrowed)

<p><b>Copyright information:</b></p><p>Taken from "Surviving extreme polar winters by desiccation: clues from Arctic springtail () EST libraries"</p><p>http://www.biomedcentral.com/1471-2164/8/475</p><p>BMC Genomics 2007;8():475-475.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2246132.</p><p></p> Accession numbers: Human AQPs 1-12A: P29972, P41181, Q92482, P55087, P55064, Q13520, O14520, O94778, O43315, Q96PS8, Q8NBQ7, Q8IXF9; Mouse AQPs 1-12A (10 is absent): Q02013, P56402, Q8R2N1, P55088, Q9WTY4, Q8C4A0, O54794, P56404, Q9JJJ3, Q8BHH1, Q8CHJ2. Bovin AQPs 1, 3, 4: P47865, Q08DE6, O77750. Dog (CANFA) AQP 1: Q9N2J4. Sheep AQP5: Q866S3. Frog AQP (RANES) (Rana esculenta): P50501. Insect AQPs: Q0IG28, Q9NHW7: AEDAE: (yellow fever mosquito); Q23808: CICVR: (green leaf hopper); Q9V5Z7: DROME () (fruit fly); Q25074: HAEIX () (buffalo fly)

Alignment of the three putative aquaporin clones identified in the libraries

<p><b>Copyright information:</b></p><p>Taken from "Surviving extreme polar winters by desiccation: clues from Arctic springtail () EST libraries"</p><p>http://www.biomedcentral.com/1471-2164/8/475</p><p>BMC Genomics 2007;8():475-475.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2246132.</p><p></p> Transmembrane domains are marked above the sequence (TM6 is only partial in both CL138 and sb_ 005_09I19). Red lines denote the two conserved NPA motifs of the aquaporin family. Only CL138 has conserved the site for N-glycosylation [31]. Red asterisks below the consensus line identify 13/14 amino acids conserved throughout the mammalian aquaporin family, as outlined in previous protein fragment analyses [59]. Percentage amino acid identities between the different springtail aquaporin clones. Figures in brackets are the percentage amino acid similarities. Each clone was clipped to the same size when performing the calculations

Example of GO molecular function (level 2) characterisation of the libraries, the data from all libraries combined is shown

<p><b>Copyright information:</b></p><p>Taken from "Surviving extreme polar winters by desiccation: clues from Arctic springtail () EST libraries"</p><p>http://www.biomedcentral.com/1471-2164/8/475</p><p>BMC Genomics 2007;8():475-475.</p><p>Published online 21 Dec 2007</p><p>PMCID:PMC2246132.</p><p></p