M. Roger Worland

How insects survive the cold: molecular mechanisms—a review

Insects vary considerably in their ability to survive low temperatures. The tractability of these organisms to experimentation has lead to considerable physiology-based work investigating both the variability between species and the actual mechanisms themselves. This has highlighted a range of strategies including freeze tolerance, freeze avoidance, protective dehydration and rapid cold hardening, which are often associated with the production of specific chemicals such as antifreezes and polyol cryoprotectants. But we are still far from identifying the critical elements behind over-wintering success and how some species can regularly survive temperatures below −20°C. Molecular biology is the most recent tool to be added to the insect physiologist’s armoury. With the public availability of the genome sequence of model insects such as Drosophila and the production of custom-made molecular resources, such as EST libraries and microarrays, we are now in a position to start dissecting the molecular mechanisms behind some of these well-characterised physiological responses. This review aims to provide a state-of-the-art snapshot of the molecular work currently being conducted into insect cold tolerance and the very interesting preliminary results from such studies, which provide great promise for the future.

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.

A method for the rapid measurement of thermal tolerance traits in studies of small insects

Abstract A technique for rapidly measuring non‐lethal thermal tolerance traits in small insects and terrestrial arthropods of similar size is described. Single or multiple individuals are heated or cooled in an arena milled into a temperature‐controlled aluminium block and their behaviour recorded continuously using a digital video camera. Data are collected retrospectively by playback of the stored images. To illustrate this technique measurement of six thermal tolerance traits using this method is described using first instar nymphs (body length = 0.66 mm) of the aphid Myzus persicae. These traits are high and low temperatures at which individual aphids cease walking, high and low temperatures at which aphids move for the final time, the temperature at which aphids begin to recover from chill coma, and the temperature at which they begin to walk again. The method is validated by comparing the results of multiple low temperature assays. No significant differences are detected between assays. Potential applications, limitations and technical problems are discussed.

The effect of feeding on specific soil algae on the cold-hardiness of two Antarctic micro-arthropods (Alaskozetes antarcticus and Cryptopygus antarcticus)

Abstract The effect of consuming terrestrial algae on the cold tolerance of two Antarctic micro-arthropods was examined. From the results of preferential feeding experiments, seven species of Antarctic terrestrial micro-algae were chosen and fed to two common, freeze-avoiding Antarctic micro-arthropods: the springtail Cryptopygus antarcticus (Collembola: Isotomidae), and the mite Alaskozetes antarcticus (Acari: Oribatida). Mites were very selective in their choice of food whereas the springtails were less discriminating. The ice nucleating activity of each species of alga was measured using an ice nucleator spectrometer and a differential scanning calorimeter. Pure cultures of individual species of algae had characteristic supercooling points ranging from ca. −5 to −18 °C. The effect of eating a particular alga on the supercooling point of individual micro-arthropods cultured at two different temperatures (0 and 10 °C) was examined. Neither species showed a preference for algae with low ice-nucleating activity and there was no clear correlation between the supercooling point of food material and that of the whole animal. However, feeding on certain algae such as Prasiola crispa, which contained the most active ice nucleators, decreased the cold tolerance of both species of arthropods.

Desiccation stress at sub-zero temperatures in polar terrestrial arthropods

Cold tolerant polar terrestrial arthropods have evolved a range of survival strategies which enable them to survive the most extreme environmental conditions (cold and drought) they are likely to encounter. Some species are classified as being freeze tolerant but the majority of those found in the Antarctic survive sub-zero temperatures by avoiding freezing by supercooling. For many arthropods, not just polar species, survival of desiccating conditions is equally important to survival of low temperatures. At sub-zero temperatures freeze avoiding arthropods are susceptible to desiccation and may lose water due to a vapour diffusion gradient between their supercooled body fluids and ice in their surroundings. This process ceases once the body fluids are frozen and so is not a problem for freeze tolerant species. This paper compares five polar arthropods, which have evolved different low temperature survival strategies, and the effects of exposure to sub-zero temperatures on their supercooling points (SCP) and water contents. The Antarctic oribatid mite (Alaskozetes antarcticus) reduced its supercooling point temperature from -6 to -30 degrees C, when exposed to decreasing sub-zero temperatures (cooled from 5 to -10 degrees C over 42 days) with little loss of body water during that period. However, Cryptopygus antarcticus, a springtail which occupies similar habitats in the Antarctic, showed a decrease in both water content and supercooling ability when exposed to the same experimental protocol. Both these Antarctic arthropods have evolved a freeze avoiding survival strategy. The Arctic springtail (Onychiurus arcticus), which is also freeze avoiding, dehydrated (from 2.4 to 0.7 g water g(-1) dry weight) at sub-zero temperatures and its SCP was lowered from c. -3 to below -15 degrees C in direct response to temperature (5 to -5.5 degrees C). In contrast, the freeze tolerant larvae of an Arctic fly (Heleomyza borealis) froze at c. -7 degrees C with little change in water content or SCP during further cold exposure and survived frozen to -60 degrees C. The partially freeze tolerant sub-Antarctic beetle Hydromedion sparsutum froze at c. -2 degrees C and is known to survive frozen to -8 degrees C. During the sub-zero temperature treatment, its water content reduced until it froze and then remained constant. The survival strategies of such freeze tolerant and freeze avoiding arthropods are discussed in relation to desiccation at sub-zero temperatures and the evolution of strategies of cold tolerance.

Survival of Heleomyza borealis (Diptera, Heleomyzidae) larvae down to − 60°C

Heleomyza borealis Boh. (Diptera, Heleomyzidae) overwinters as larvae in Arctic habitats, where they may experience winter temperatures below − 15°C. The larvae freeze at c.− 7°C but in acclimation experiments 80% survived when exposed to − 60°C. Of the larvae exposed to between − 4 and − 15°C, only 3% pupated. However, when cooled to − 20°C this increased to 44%, with 4% emerging as adults. Larvae maintained at 5°C contained low levels of glycerol, sorbitol and trehalose, which did not increase with acclimation to low temperatures. However, levels of fructose increased from 6.1 μg mg−1 fw in control animals to 17 μg mg−1 fw when exposed to − 2°C for 1 week. Larval body water (2.2 ± 0.1 g/g dw, mean ± SD, n = 100) and lipid content (0.22 ± 0.002 g/g fw, mean ± SE) showed no significant change during acclimation to low temperatures. Larvae maintained at a constant 5°C survived for over 18 months with little loss of body mass (from 7.5 ± 1.2 to 7.0 ± 1.2 mg fw, mean ± SD, n = 20), but none pupated. Heleomyza borealis larvae appear to feed and grow until they reach a body mass of about 7.5 mg and then become dormant. They remain in this state until they experience a low temperature stimulus (< − 15°C) followed by a warm period (≈ 5°C). This ensures that the larvae pupate and adults emerge in early summer, allowing the maximum growing period before the following winter. Heleomyza borealis are adapted to survive long winters in a dormant larval state. They have a low metabolic rate, can conserve body water even at subzero temperatures but do not synthesize large quantities of cryoprotectants.

Spatial distribution, habitat preference and colonization status of two alien terrestrial invertebrate species in Antarctica

Abstract The introduction of invasive species is one of the greatest threats to Earth’s biodiversity, as they can reduce native biodiversity and alter ecosystem structure and function. Currently, the only two known non-native terrestrial invertebrates in Antarctica are the chironomid midge Eretmoptera murphyi and the enchytraeid worm Christensenidrilus blocki. These invertebrates were probably introduced to ground near Signy Research Station, South Orkney Islands, during transplantation experiments in the late 1960s. Between 2007 and 2009, this study surveyed the area around the introduction site for midge larvae and worms to assess any change over the last four decades in their spatial distribution, habitat preference and colonization status. Eretmoptera murphyi was found in concentrations up to 4.1 × 105 larvae m-2 (mean 2.1 × 104 larvae m-2) at distances of up to 220 m from the probable introduction site (c. 35 000 m2), while C. blocki was only found close to the introduction site in low numbers. Significantly more E. murphyi larvae were found in peat and dead organic material (3.34 × 104 m-2) than in stony soil and gravel (1.52 × 104 m-2) or living moss and other plant material (1.16 × 104 m-2). Eretmoptera murphyi can no longer be considered a persistent alien as it clearly expanding its distribution, while C. blocki remains a persistent alien species.

Ice nucleation and freezing tolerance in New Zealand alpine and lowland weta, Hemideina spp. (Orthoptera; Stenopelmatidae)

Abstract.The alpine tree weta Hemidiena maori Pictet et Saussure (Orthoptera: Stenopelmatidae) is a large, flightless insect found above the treeline on many of the mountain ranges of the South Island of New Zealand. The population found on the Rock and Pillar Range, Central Otago has been identified as freezing tolerant with a haemolymph ice nucleating agent. The ability of H. maori to survive freezing is compared to the lowland weta Hemideina thoracica Walker and H. crassidens Blanchard, both of which are able to survive the formation of some ice in their bodies. Mortality is associated with time spent frozen in H. thoracica, and it is hypothesized that this species is killed when a critical proportion of its body water is frozen. All five subalpine and alpine populations of H. maori surveyed were found to be freezing tolerant.

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.

Primary succession of lichen and bryophyte communities following glacial recession on Signy Island, South Orkney Islands, Maritime Antarctic

Abstract A directional primary succession with moderate species replacement was quantitatively characterized on Signy Island in zones of a glacial valley corresponding to their age since deglaciation. A continuous increase in diversity and abundance of lichens and bryophytes was observed between terrains deglaciated in the late 20th century, to areas where deglaciation followed the Little Ice Age, and others thought to be ice-free since soon after the Last Glacial Maximum. Classification (UPGMA) and ordination (principal co-ordinate analysis) of vegetation data identified three different stages of development: a) pioneer communities, which rapidly develop in a few decades, b) immature communities developing on three to four century old terrains, and c) a climax stage (Polytrichum strictum-Chorisodontium aciphyllum community) developing on the oldest terrains, but only where local-scale environmental features are more favourable. Multivariate analysis including environmental parameters (canonical correspondence analysis) indicated terrain age as being the dominant controlling factor, with other environmental factors also exhibiting significant conditional effects (duration of snow cover, surface stoniness). These findings not only quantitatively verify reports of the rapid colonization of Maritime Antarctic terrains following recent climate amelioration and associated decrease in glacial extent, but also show how local-scale environmental resistance may slow or even prevent vegetation succession from pioneer to more mature stages in future.