Gavin Burns

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

BackgroundInsects 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 Megaphorura arctica, formerly Onychiurus arcticus (Tullberg 1876). The physiological mechanisms underlying cryoprotective dehydration have been well characterised in M. arctica and to date this process has been described in only a few other species: the Antarctic nematode Panagrolaimus davidi, an enchytraied worm, the larvae of the Antarctic midge Belgica antarctica and the cocoons of the earthworm Dendrobaena octaedra. There are no in-depth molecular studies on the underlying cold survival mechanisms in any species.ResultsA cDNA microarray was generated using 6,912 M. arctica 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 M. arctica 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.ConclusionMicroarray 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.

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 SCPs.

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.

Transcriptome pyrosequencing of the Antarctic brittle star Ophionotus victoriae

Brittle stars are included within a whole range of species, which contribute to knowledge in the medically important area of tissue regeneration. All brittle stars regenerate lose limbs, but the rate at which this occurs is highly variable and species-specific. One of the slowest rates of arm regeneration reported so far is that of the Antarctic Ophionotus victoriae. Additionally, O. victoriae also has an unusual delay in the onset of regeneration of about 5months. Both processes are of interest for the areas of regeneration biology and adaptation to cold environments. One method of understanding the details of regeneration events in brittle stars is to characterise the genes involved. In the largest transcriptome study of any ophiuroid to date, we describe the results of mRNA pyrosequencing from pooled samples of regenerating arms of O. victoriae. The sequencing reads resulted in 18,000 assembled contiguous sequences of which 19% were putatively annotated by blast sequence similarity searching. We focus on the identification of major gene families and pathways with potential relevance to the regenerative processes including the Wnt/β-catenin pathway, Hox genes, the SOX gene family and the TGF beta signalling pathways. These data significantly increase the amount of ophiuroid sequences publicly available and provide candidate transcripts for the further investigation of the unusual regenerative process in this Antarctic ophiuroid.

Characterisation of the warm acclimated protein gene (wap65) in the Antarctic plunderfish (Harpagifer antarcticus)

Physiological adaptation to increased environmental temperatures has been studied experimentally in a number of fish species, with the up-regulation of several genes identified as being associated with the process, such as the warm-acclimated protein (wap-65). This article describes the cloning and characterisation of the wap65-2 gene from the Antarctic plunderfish (Harpagifer antarcticus). The transcriptional expression of this gene in response to elevated seawater temperatures over a time course series is presented. Initially there is strong down-regulation of this gene to a maximum of 40-fold within 4 h, followed by recovery to almost control levels within 48 h, indicating that this gene does not play a role in the potential temperature adaptation of H. antarcticus.