Surviving extreme polar winters by desiccation: clues from Arctic springtail (Onychiurus arcticus) EST libraries
© Clark et al; licensee BioMed Central Ltd. 2007
Received: 06 September 2007
Accepted: 21 December 2007
Published: 21 December 2007
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.
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.
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.
The mechanisms by which organisms survive extreme low temperatures are not only of interest to ecologists, but also to a number of applied medical fields . In this respect, one of the most amenable and studied groups of organisms is the Collembola (arthropods or springtails) where the physiological processes behind such survival are well documented [2, 3]. They possess three main strategies to survive the cold: freeze tolerance, freeze avoidance or protective dehydration [3–5]. Whilst most springtails use freeze avoidance, it is the latter strategy of protective dehydration in the Arctic springtail Onychiurus arcticus (Tullberg 1876) which is the subject of this study [5, 6]. In protective dehydration loss of water occurs across a diffusion gradient between the animal's super-cooled body fluids and ice in its surroundings, such that freezing point depression always exceeds the environmental temperature experienced, and eventually the animals lose sufficient water to ensure that a freezing event cannot occur [5, 6]; the animals desiccate. O. arcticus is widely distributed throughout the northern parts of the Palaearctic region [7–10] and is found in moist habitats; mainly in mosses and under large stones in the coastal areas of Svalbard, particularly on glacial outwash fans and under bird cliffs . Studies have shown that O. arcticus, exposed to sub-zero temperatures and low water vapor pressure induces extensive dehydration through a highly permeable cuticle [5, 12, 13]. This is combined with the rapid synthesis and accumulation of the membrane/protein cryoprotectant trehalose from glycogen [13–15].
Whilst there are a number of physiological and ecological studies on this organism (detailed above), there have been no molecular analyses to date (only O. groenlandicus has been bar-coded: AY665335, AY6653316, AY665323). This situation is not unusual, as the number of organisms where there is even moderate amounts of sequence data are severely limited. However, genomics is being increasingly applied to the study of non-model organisms and ESTs are generally viewed as the most efficient and cost effective strategy for the identification of genes and generating a first pass scan of a genome [16, 17].
As part of a larger project examining over-wintering strategies in polar arthropods, we have generated 16,379 ESTs for O. arcticus from 5 cDNA libraries of animals in different desiccation states. In this article we present the analysis of these library data. This represents the first sequence data for this organism and significantly increases the number of Collembolan ESTs in the databases from the previous total of 8,686 produced from the springtail Folsomia candida .
Results and Discussion
CDNA libraries construction and characterisation
Five libraries were produced from both desiccating and re-hydrating populations of O. arcticus from stages that had previously been defined as potentially informative for molecular analyses .
• Library C: Controls
• Library D1: Desiccating: animals at -2°C. This is the critical temperature at which trehalose is significantly up-regulated at the expense of stores of glycogen.
• Library D2: Fully desiccated animals.
• Library R1: Animals that had been recovering for 8 hours.
• Library R2: Fully recovered animals.
Recovering 8 hr
Recovered 24 hr
# clusters (> 1 read)
# putative transcripts
Average cluster size
Largest cluster (# reads)
# clusters with 2 ESTs
# clusters with 3 ESTs
# clusters with 4–5 ESTs
# clusters with 6–10 ESTs
# clusters with > 10 ESTs
#(%) with significant **Swissprot hits
#(%) with significant **trembl hits
#(%) with no hits
The decision was made to analyse the libraries using GO annotation rather than keyword extraction from Blast comment lines, as GO categories are more generic than gene names. This facilitates the comprehensive identification of gene sets associated with biochemical pathways without in-depth knowledge of individual pathway components and also where potentially multiple pathways exist for a particular process (cf. trehalose ). In the following discussion PGO refers to a Biological Process GO annotation; FGO is Molecular Function and CGO is Cellular component.
Initially the focus of the searches concentrated on general processes such as response to water, water deprivation, abiotic stimulus, desiccation and Qdrought recovery. Disappointingly only 11 clones were identified from the five libraries, but even so, these did include some clones of further interest. PGO:0009414, response to water deprivation identified a putative aquaporin (the same clones were also identified under a specific GO search for aquaporin) and PGO:0009415, response to water, produced ESTs with matches to dehydrin which is induced in response to water stress in plants, the latter of which would not have been identified using extraction of Blast keyword data. Given the relative lack of success of the generic searches, specific genes and pathways were targeted. Of the genes present in the libraries, there was particular interest in identifying the following genes:
• The trehalose and glycogen pathways (as trehalose is produced from glycogen during the desiccation process) .
• Genes involved in cell protection, such as antioxidants
• LEA (Late Embryogeneis Abundant) proteins, which have been shown to be involved in desiccation in a number of organisms .
Trehalose and glycogen pathways
alpha, alpha-trehalase activity; FGO:0004555
trehalase activity; FGO:0015927
alpha, alpha-trehalose-phosphate synthase (UDP-forming) activity; FGO:0003825
alpha, alpha-trehalose-phosphate synthase complex (UDP-forming); CGO:0005946
trehalose biosynthetic process; PGO:0005992
trehalose catabolic process; PGO:0005993
Trehalose metabolic process; PGO:0005991
Trehalose transmembrane transporter activity; FGO:0015574
trehalose transport; PGO:0015771
Trehalose-phosphatase activity; FGO:0004805
Trehalose 6 phosphate synthase
Spodoptera exigua (beet armyworm)
FGO:0003825 FGO:0004805 PGO:0005991
LATS tumour suppressor
Serine/threonine protein kinase 38
Aedes aegypti (yellow fever mosquito)
Protein kinase A camp dependant catalytic subunit
Artemia sanfranciscana (brine shrimp)
P70 ribosomal protein S6 kinase
Putative protein kinase DC2
Similar to serine/threonine protein kinase 6 (Aurora family kinase 1)
Apis mellifera European honey bee
Protein kinase C
CAMP dependant protein kinase C1
Bombyx mori (silk moth)
These proteins are associated with water transport across membranes (Kruse et al, 2006) and have the GO annotation: FGO:0015250: water channel activity. Searches of all libraries revealed 7 clones in total (4 singletons and a cluster of 3) matching three potentially different aquaporin genes:
• Q0IG28: Aquaporin 1 from Aedes aegypti. Two clones from control and desiccating libraries (C and D1), matches in excess of P value = 177, expect value = 9.7e-12.
• Q9NHW7: Aquaporin AQPAe.a from Aedes aegypti. Two clones from desiccated and recovering libraries (D2 and R2), matches in excess of P value = 512, expect value = 3.2 e-47
• UniRef100 000051A00B: Predicted similar to Drip CG9023_PB isoform B Apis mellifera. 1 cluster of 3 genes from the desiccated library (D2) with a match of P value = 480, expect value = 7.7 e-44
Number of clones
Number of clones
Number of clones
Number of clones
Number of clones
Number of clones
Number of clones
Processes involved in the survival of insects at low temperatures include the removal or deactivation of ice nucleating agents, accumulation of cryoprotectants and thermal hysteresis proteins [35–38]. Moulting has recently been shown to be associated with reduction of the supercooling point (SCP) and hence cryoprotection in Antarctic springtails (Crypotpygus antarcticus) [28, 29]. This might be expected to depress the SCP, because in Collembola the mid-gut and its entire contents are shed during moulting  resulting in the expulsion of potential ice nucleators in the animal gut. The physiology and timing of moulting in O. arcticu s has not been documented to date, although with a rigid exoskeleton they clearly moult regularly in order to grow. So genes and pathways involved in moulting were also investigated using GO annotations (data not shown). A number of relevant genes were identified for both juvenile hormone (a pleiotropic hormone, which in concert with ecdysteroids orchestrates moulting and metamorphosis and may be involved in reproduction in some species) and members of the ecdysone pathway. The latter included the ecdysone receptor and the protein ultraspiracle (XR2C) chorion factor. In addition to a considerable number of putative transcription factors and chromatin remodelling subunits were identified. It was interesting to note that more matches were found in the actively desiccating (D1) and recovering animals (LIBs R1 and R2) (10, 24 and 14 respectively, total = 48) compared to only 8 in the control and 5 in the desiccated populations. Thus indicating that moulting may either play a role in the desiccation process, or potentially is triggered in some animals by the cellular stress involved in desiccation and recovery. We are currently investigating the role of moulting in desiccation of O. arcticus using a biochemical test for 20-hydroxy ecdysone and are also actively collecting animals that have either just moulted or are in the process of moulting for more detailed molecular analysis.
Database searches of LEA proteins can produce matches to abhydrolase genes (as happened with this clone), due to the presence of an abhydrolase domain (denoted in Figure 4). Abhydrolases are a largely uncharacterised protein family, but members of this family do contain domains with hydrolase activity and therefore could potentially be involved in desiccation biochemistry. Phylogenetic analysis of the O. arcticus translated gene fragment suggests that this fragment most closely matches the ABHD_A (or ABHD_10) abhydrolase domain (data not shown). The question remains, is this clone sb_009_02E03 an LEA or an abhydrolase? This is virtually impossible to answer with short sequence fragments and the answer may become apparent with the full-length sequence, the cloning of which by RACE PCR is now underway. Also Western blotting studies are being carried out on O. arcticus using heterologous probing of a LEA antibody to identify such proteins via an alternative route.
Differences between the libraries
RECOVERED (24 hrs)
Putative LEA protein
• Aquaporin 1
• Aquaporin AQPAE.a
• Aquaporin CG9023
This paper describes, for the first time, EST data from the arctic springtail (O. arcticus), significantly enhancing the number of Collembolan ESTs in the public databases. 40% of the clones produced significant matches against Swissprot and trembl and these ESTs were further analysed using GO annotations. This facilitated the identification of genes involved in biochemical pathways of interest, such as trehalose biosynthesis and moulting. The GO annotations produced a greater range of potential "genes" for further investigation and was more effective at identifying genes in a particular pathway than could have been identified using extraction of data from Blast. Candidate genes involved in the desiccation process were identified including three members of the aquaporin family and a putative LEA protein. These genes are under further investigation. The GO annotations identified in this publication will be used to automatically extract EST clone ids from in-house produced insect libraries to target further investigations into over-wintering survival of insects in extreme environments. This will include construction of customised microarrays.
Sample collection and preparation
Onychiurus arcticus, were collected under the bird cliffs at Stuphallet and Krykkefjellet on the Brøggerhalvøya, near Ny Ålesund, Spitsbergen, Svalbard, Norway (78°56'N, 11°53'E) and transported to the British Antarctic Survey (BAS), Cambridge, for analysis. Animals (mixture of both adult and juveniles) were cultured in ventilated plastic boxes containing moss, lichen and soil taken from field sites and fed on dried baker's yeast. Cultures were kept moist at +4°C.
Five groups of animals were prepared for library production:
• Library C: Controls: live animals which were kept in a +4°C cabinet
• Library D1: Desiccating: animals were cooled for two weeks from +2 to -2°C in culture pots containing a base of wet plaster of Paris/charcoal at a rate of 2°C per week. -2°C is the critical temperature at which trehalose is significantly up-regulated at the expense of stores of glycerol.
• Library D2: Fully Desiccated: animals were cooled from +2 to -14°C in culture pots containing a base of wet plaster of Paris/charcoal at a rate of 2°C per week.
• Library R1: Recovering: animals from the -14°C group were allowed to recover at +5°C with moisture for 8 hours.
• Library R2: Fully recovered: animals from the -14°C group were allowed to recover at +5°C moisture for 24 hours.
All groups of animals were rapidly frozen in liquid nitrogen and kept at -80°C until required.
Total RNA was extracted from frozen (-80°C) samples of O. arcticus using TRI Reagent (Sigma) according to manufacturer's instructions. cDNA was synthesized and normalized using a combination of the Trimmer-Direct kit (Evrogen, Moscow, Russia) and the SMART cDNA Library Construction kit (BD Biosciences Clontech, Palo-Alto, CA) according to the protocol from Evrogen . Modifications to the protocol were made concerning the columns used for size selection and the cloning vector: to improve clone size selection Chroma spin 1000 were used instead of Chroma spin 400 (both BD Biosciences Clontech, Palo-Alto, CA) and pal32 (Evrogen, Moscow, Russia) was used for directional cloning with insertion between two SfiI sites (GGCCATTACGGCCGGG del(CATGTC) GGCCGCCTCGGCC. This procedure was chosen because of the low amount of starting material  and the normalisation process increased the efficiency of rare transcript discovery [42, 43]. Plasmids were transferred via electroporation to Escherichia coli (strain DH10B, Invitrogen, Karlsruhe, Germany).
Plasmids were isolated according to the method of  and 5'end sequenced using Dye Terminator Chemistry version 3.1 (ABI, Weiterstadt, Germany) and 3730XL ABI capillary sequencer systems (ABI, Weiterstadt, Germany).
Sequence analysis and EST clustering
Sequence fasta files were processed using the script Trace2dbest , which incorporated the phred [46, 47] and crossmatch (P. Green, unpublished) programmes. A minimum cut-off value of 150 bp was applied after quality control processing for sequence database searching and for generating the submission file for dbEST  (Accession numbers, dbEST: 49109381–49125759, Genbank: EW744731–EW761109). Tgicl  was used for clustering the fasta files, incorporating quality scores, for each of the five libraries, as well as for all the libraries together. The clusters were database searched using Blastx  against the Uniprot/Swissprot and Uniprot/Trembl databases  (at 12/06/2007), with matches annotated for all scores with an expect score in excess of 1e-10. Sequences with a database match were then further annotated using GO  (at 24/07/2007). Another view of the data was generated by Blast2GO  using the non-redundant (nr) database . With the Blast2Go annotation, the programme GOSSIP  was run to identify any enrichment for GO annotations between the libraries. Sequence manipulation was carried out using the EMBOSS suite of programmes . Sequences were clustered using ClustalW  and then subjected to phylogenetic analysis using the Phylip suite of programmes with bootstrapping  and displayed using MEGA4 . Sequence alignments were displayed using BoxShade v3.21 .
This paper was produced within the BAS Q4 BIOREACH/BIOFLAME core programmes and also contributes to the SCAR EBA programme. JP was sponsored by the EU Sleeping Beauty Consortium: Specific Targeted Research Project, Contract no 012674 (NEST). JP and GG-L are also funded by the MSTD grant 143034, awarded by the Republic of Serbia. The authors would like to thank the NEBC Bio-linux team http://nebc.nox.ac.uk/ for assistance with the bioinformatics and provision of software packages. Also NERC for yearly access to the NERC Arctic Research Station (Harland Huset) at Ny-Ålesund and Nick Cox, the Arctic base commander.
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