Gordana Grubor-Lajšić

Partial desiccation induced by sub-zero temperatures as a component of the survival strategy of the Arctic collembolan Onychiurus arcticus (Tullberg)

The mechanism by which the freeze susceptible Arctic collembolan Onychiurus arcticus survives winter temperatures of -25 degrees C in the field is not fully understood but exposure to sub-zero temperatures (e.g. -2.5 degrees C) is known to induce dehydration and lower the supercooling point (SCP). In this study, changes in the water status and certain biochemical parameters (measured in individual Collembola) during a 3-week exposure to decreasing temperatures from 0 to -5.5 degrees C were studied. Osmotically active and inactive body water contents were measured by differential scanning calorimetry (DSC), water soluble carbohydrates by high performances liquid chromatography (HPLC) and glycogen by enzymatic assays. The activity of trehalase and trehalose 6-phosphate synthase were also measured. During the experiment, total water content decreased from 70 to 40% of fresh weight, mostly by the loss of osmotically active water with only a small reduction in the osmotically inactive component. The SCP decreased from -7 to -17 degrees C. Analysis of the results shows that if O. arcticus is exposed to -7 degrees C in the presence of ice, all osmotically active water would be lost due to the vapour pressure gradient between the animals supercooled body fluids and the ice. Under these conditions the estimated SCP would reach a minimum of c. -27 degrees C, but the Collembola may never freeze as all the osmotically active water has been lost, the animal becoming almost anhydrobiotic. Trehalose concentration increased from 0.9 to 94.7&mgr;g mg(-1)fw while glycogen reserves declined from 160 to 7.7 nmol glucose equivalents mg(-1) protein. Trehalase activity declined as the temperature was reduced, while trehalose 6-phosphate activity peaked at 0 degrees C. By adopting a strategy of near anhydrobiosis induced by sub-zero temperatures, O. arcticus, which was previously thought to be poorly adapted to survive severe winter temperatures, is able to colonise high Arctic habitats.

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.

Comparison of the cold hardiness of two larval Lepidoptera (Noctuidae)

Abstract Diapause larvae of the European corn borer (Ostrinia nubilalis (Hubn.)) and the related Mediterranean noctuid Sesamia cretica Led. possess sufficient supercooling ability to avoid freezing over their normal environmental temperature ranges. In progressive chilling experiments (10 days acclimation at each 5° step in the temperature range from 15 to −5°C), mean supercooling points (measured at a cooling rate of 0.1°C min−1) were lowered from −20.4°C at 15°C to −24.0°C at 5°C (lower lethal temperatures: c.−28°C) in O.nubilalis, compared with −15.0 to −17.2°C (lower lethal temperatures: −15 to −17°C respectively) in S.cretica. Concentrations of glycerol and trehalose determined by gas chromatography of whole body extracts were consistently higher in the former than in the latter species at both 15 and 5°C, and may be responsible for the deeper supercooling in O.nubilalis larvae. Acclimation to 5°C increased glycerol levels in O. nubilalis extracts compared with 15°C, and this was enhanced in larvae exposed for a further 10 days at each of 0 and −5°C (glycerol being 438μmol ml−1 body water). Haemolymph glycerol concentrations showed a similar pattern to whole body extracts in this species. Fat body glycogen was reduced during low temperature acclimation in both species. Body water contents did not change with acclimation in O.nubilalis, whilst S.cretica, containing significantly more water, lost c.7% during acclimation from 15 to 5°C. Haemolymph osmolalities increased during acclimation, especially in Ostrinia larvae, probably as a result of the accumulation of cryoprotectants. The majority of O.nubilalis larvae survived freezing under the conditions of the cooling experiments, whilst larvae of S.cretica did not, thereby confirming an element of freezing tolerance in the former.

Expression of stress-related genes in diapause of European corn borer (Ostrinia nubilalis Hbn.).

Diapause is a state of arrested development during which insects cope with many external and internal stressful factors. European corn borer, Ostrinia nubilalis, overwinters as a fifth instar freeze-tolerant diapausing larva. In order to explore diapause-linked stress tolerance processes, the expression of selected genes coding for stress-related proteins-glutathione S-transferase (Gst), thioredoxin (Trx), glutaredoxin (Grx), ferritin (Fer), metallothionein (Mtn), and heat shock proteins Hsp90, Hsc70, Hsp20.4, and Hsp20.1-was assessed in the fat body of diapause-destined, warm (22 °C) and cold (5 °C) acclimated diapausing larvae using the quantitative real-time PCR. Gene expression was normalised to mRNA transcripts for Actin and Rps03, and relative expression was calculated using non-diapausing larvae as a control group. During the initiation phase of diapause, the abundance of mRNA transcripts of Grx, Hsp90, Hsc70, and Hsp20.1 was significantly upregulated, Trx, Fer, Mtn, and Hsp20.1 were unchanged, while only Gst was clearly downregulated in comparison to non-diapause control. Later, in the early phase of diapause, the expression of most genes (except Trx and Hsp20.1) was upregulated in warm-acclimated larvae, while only Trx and Hsp90 were upregulated in cold-acclimated larvae. Furthermore, the relative expression of all genes (except Trx) increased gradually throughout the diapause in cold-acclimated larvae. This result indicates that the half-life of mRNAs is prolonged during diapause at low temperature, which may lead to a gradual accumulation of mRNA transcripts. Our results show that both diapause programming and temperatures affect the expression of stress-related genes in Ostrinia nubilalis.

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.