Jelena Purać

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.

Importance of the Body Water Management for Winter Cold Survival of the European Corn Borer Ostrinia Nubilalis Hübn. (Lepidoptera: Pyralidae)

ABSTRACT Winter diapause, a common strategy of many insect species occupying temperate regions, is usually closely related to and coincides with their cold hardiness. Freezing of body fluids represents one of the major obstacles for sub-zero temperatures survival and thus the body water management is an important part of cold hardiness. In this study, we examined some cryobiological parameters, as well as content of glycerol and trehalose in non-diapausing and freeze tolerant diapausing larvae of the European corn borer, Ostrinia nubilalis. Diapausing larvae were divided into two experimental groups—a group exposed to field temperatures (which were in average above 0°C) and a group exposed to -8°C for ten days. Contents of the total body water, osmotically active (OA) and inactive (OI), as well as the supercooling point (SCP) of hemolymph and fat body, were measured by differential scanning calorimetry (DSC). The content of glycerol and trehalose was analysed by gas chromatography. Compared to diapausing groups, non -diapausing larvae had higher SCP, lower content of trehalose and glycerol in both tissues. The content of total and OA water in both tissues of diapausing larvae had changed with low temperatures exposure. At -8°C, the amount of total and OA body water was decreased in hemolymph and increased in fat body while the content of OI water was slightly increased in hemolymph but remained unchanged in fat body. Mean SCPs of both tissues were significantly different—for hemolymph it was around -21°C, which was almost two times lower than for fat body (-10°C). However, the SCPs of fat body and hemolymph had not significantly changed after the exposure to low temperature. The content of glycerol and trehalose was far greater in hemolymph than in fat body for all groups, which is in accordance with the difference between the SCPs of these tissues. Furthermore, exposure of diapausing larvae to sub-zero temperatures (-8°C) had simultaneously provoked an increase in glycerol/trehalose concentration in hemolymph and the decrease in fat body. These adjustments of water and cryoprotectors distribution are an important part of cold hardiness mechanisms.

Cold hardening induces transfer of fatty acids between polar and nonpolar lipid pools in the Arctic collembollan Megaphorura arctica

Cold hardiness in the Arctic Collembola Megaphorura arctica (Tullberg), formerly Onychiurus arcticus, has been the subject of extensive studies over the last decade. This species employs an unusual strategy known as cryoprotective dehydration to survive winter temperatures as low as −25 °C. To expand knowledge of cryoprotective dehydration in M. arctica, the present study investigates how a reduction in ambient temperature affects the fatty acid composition of the total body lipid content along with polar (mainly membrane phospholipids) and nonpolar (mainly triacylglycerols) lipids. Most ectothermic animals compensate for changes in fluidity by regulating fatty acid composition, a process often described as homeoviscous adaptation. In M. arctica, changes in the fatty acid composition of total body lipid content during cold treatment are only moderate, with no clear pattern emerging. However, the levels of unsaturated fatty acids in the polar lipids increase with cold exposure, largely attributable to 16 : 1(n− 7), 18 : 1(n− 9), 18 : 3(n− 6) and 18 : 3(n− 3), whereas unsaturated fatty acid levels in the nonpolar lipids correspondingly decrease. These results suggest a reallocation of fatty acids between the two lipid pools as a response to a temperature reduction of 6 °C. Because of hypometabolism, a characteristic of cold adaptation, such a mechanism could be less energy demanding than de novo synthesis of fatty acids and may comprise part of an adaptive homeostatic response.

Diapause induces remodeling of the fatty acid composition of membrane and storage lipids in overwintering larvae of Ostrinia nubilalis, Hubn. (Lepidoptera: Crambidae).

Seasonal changes in the FA composition of triacylglycerols and phospholipids prepared from the whole bodies of non-diapausing and diapausing fifth instar larvae of Ostrinia nubilalis, Hubn. (Lepidoptera: Crambidae) were determined to evaluate the role of these lipids in diapause. Substantial changes in the FA composition of triacylglycerols and phospholipids were triggered by diapause development. This led to a significant increase in the overall FA unsaturation (UFAs/SFAs ratio), attributable to an increase in the relative proportion of MUFAs and the concomitant decrease in PUFAs and SFAs. In triacylglycerols, the significant changes in the FAs composition are the result of an increase in the relative proportions of MUFAs, palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), and a concomitant reduction in the composition of SFAs and PUFAs, mainly palmitic acid (16:0) and linoleic acid (18:2n-6), respectively. Changes in the composition of phospholipids were more subtle with FAs contributing to the overall increase of FA unsaturation. Differential scanning calorimetry (DSC) analysis revealed that the melt transition temperatures of total lipids prepared from whole larvae, primarily attributable to the triacylglycerol component, were significantly lower during the time course of diapause compared with non-diapause. These observations were correlated to the FA composition of triacylglycerols, most likely enabling them to remain functional during colder winter conditions. We conclude that O. nubilalis undergoes remodeling of FA profiles of both energy storage triacylglycerols and membrane phospholipids as an element of its overwintering physiology which may improve the ability to cold harden during diapause.

Diapause induces changes in the composition and biophysical properties of lipids in larvae of the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae)

This study compares the composition and biophysical properties of lipids in non-diapausing and diapausing fifth instar larvae of Ostrinia nubilalis Hubn. (Lepidoptera: Crambidae). The majority of fat body lipids in both of these physiological states were comprised of ~90% triacylglycerols (TAGs), whereas the haemolymph contained a more even distribution of all lipid classes. The fatty acid composition and biophysical properties of the fat body lipids differed markedly between non-diapausing and diapausing larvae. Diapause was associated with a dramatic increase in the proportions of palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), with concurrent reductions in palmitic acid (16:0) and linoleic acid (18:2n-6). The increase in the level of unsaturation of the fat body lipids, which caused a marked shift in their phase transitions to lower temperatures, was triggered by diapause rather than low temperatures. Adjustments of fatty acid compositions are likely to be an important component of winter diapause mechanisms, possibly maintaining the fluidity of cell membranes and the functionality of the organism during lower winter temperatures.

Cold Adaptation Responses in Insects and Other Arthropods: An “Omics” Approach

In this chapter, we review recent genomic, proteomic, and metabolomic studies that link several gene and protein products involved in cold adaptation in insects and other arthropods to energy metabolism and cellular protection mechanisms. Organisms have evolved various mechanisms for survival at subfreezing temperatures. In general, cold hardy invertebrates utilize four main strategies to survive cold temperatures: (1) freeze tolerance, (2) freeze avoidance, (3) cryoprotective dehydration, and (4) vitrification. In addition, many insects in temperate regions overwinter in an arrested developmental state known as diapause, during which they are cold hardy. Major alterations occur during winter diapause, with respect to both total metabolic flux and the relative activities of different metabolic pathways. In these organisms, one such metabolic adaptation to unfavorably cold environmental conditions is the synthesis of cryoprotectants/anhydroprotectants. The metabolic changes and metabolic paths involved in cold adaptation suggest involvement of specific enzymes and key regulatory proteins. These mechanisms of cold adaptation require precise scheduling of the expression of specific genes. Thus, we discuss here the evidence researchers have recently begun to gather supporting a relationship between the genes and proteins of the cold adaptation response and mechanisms of cellular protection and energy metabolism using an “omics” approach.

HYDROGEN PEROXIDE AND ECDYSONE IN THE CRYOPROTECTIVE DEHYDRATION STRATEGY OF Megaphorura Arctica (ONYCHIURIDAE: COLLEMBOLA)

The Arctic springtail, Megaphorura arctica, survives sub-zero temperatures in a dehydrated state via trehalose-dependent cryoprotective dehydration. Regulation of trehalose biosynthesis is complex; based in part on studies in yeast and fungi, its connection with oxidative stress caused by exposure of cells to oxidants, such as hydrogen peroxide (H₂O₂), or dehydration, is well documented. In this respect, we measured the amount of H₂O₂ and antioxidant enzyme activities (superoxide dismutases: copper, zinc--CuZnSOD and manganese containing--MnSOD, and catalase--CAT), as the regulatory components determining H₂O₂ concentrations, in Arctic springtails incubated at 5 °C (control) versus -2 °C (threshold temperature for trehalose biosynthesis). Because ecdysone also stimulates trehalose production in insects and regulates the expression of genes involved in redox homeostasis and antioxidant protection in Drosophila, we measured the levels of the active physiological form of ecdysone--20-hydroxyecdysone (20-HE). Significantly elevated H₂O₂ and 20-HE levels were observed in M. arctica incubated at -2 °C, supporting a link between ecdysone, H₂O₂, and trehalose levels during cryoprotective dehydration. CAT activity was found to be significantly lower in M. arctica incubated at -2 °C versus 5 °C, suggesting reduced H₂O₂ breakdown. Furthermore, measurement of the free radical composition in Arctic springtails incubated at 5 °C (controls) versus -2 °C by Electron Paramagnetic Resonance spectroscopy revealed melanin-derived free radicals at -2 °C, perhaps an additional source of H₂O₂. Our results suggest that H₂O₂ and ecdysone play important roles in the cryoprotective dehydration process in M. arctica, linked with the regulation of trehalose biosynthesis.

ENVIRONMENTAL EFFECTS ON SUPEROXIDE DISMUTASE AND CATALASE ACTIVITY AND EXPRESSION IN HONEY BEE

Understanding the cellular stress response in honey bees will significantly contribute to their conservation. The aim of this study was to analyze the response of the antioxidative enzymes superoxide dismutase and catalase in honey bees related to the presence of toxic metals in different habitats. Three locations were selected: (i) Tunovo on the mountain Golija, as control area, without industry and large human impact, (ii) Belgrade as urban area, and (iii) Zajača, as mining and industrial zone. Our results showed that the concentrations of lead (Pb) in whole body of bees vary according to habitat, but there was very significant increase of Pb in bees from investigated industrial area. Bees from urban and industrial area had increased expression of both Sod1 and Cat genes, suggesting adaptation to increased oxidative stress. However, in spite increased gene expression, the enzyme activity of catalase was lower in bees from industrial area suggesting inhibitory effect of Pb on catalase.

Lea protein expression during cold-induced dehydration in the Arctic Collembola Megaphorura arctica

The Arctic springtail Megaphorura arctica (Tullberg, 1876) employs a strategy known as cryoprotective dehydration to survive winter temperatures as low as -25 degrees C. During cryoprotective dehydration, water is lost from the animal to ice in its surroundings as a result of the difference in vapour pressure between the animals supercooled body fluids and ice (Worland et al., 1998; Holmstrup and Somme, 1998). This mechanism ensures that as the habitat temperature falls, the concentration of solutes remains high enough to prevent freezing (Holmstrup et al., 2002). In M. arctica, accumulation of trehalose, a cryo/anhydro protectant, occurs in parallel with dehydration. Recent studies have identified a number of genes and cellular processes involved in cryoprotective dehydration in M. arctica (Clark et al., 2007; Clark et al., 2009; Purac et al., 2011). One of them includes late embryogenesis abundant (LEA) proteins. This study, together with that of Bahrndorff et al. (2008), suggests that LEA proteins may be involved in protective dehydration in this species.