Temperature adaptation of lipids in diapausing Ostrinia nubilalis: an experimental study to distinguish environmental versus endogenous controls.

Larvae of the European corn borer (Ostrinia nubilalis Hubn.) were cold acclimated during different phases of diapause to determine if changes in the fatty acid composition lipids occur as part of a programmed diapause strategy, or as a response to low temperatures during winter. Cold acclimation of fifth instar larvae of O. nubilalis during diapause had modest effects further on the readjustments in fatty acid composition of triacylglycerols and phospholipids. Overall, FA unsaturation (UFAs/SFAs ratio) was stable, with the exception of the triacylglycerols fraction after exposure to -3 and -10 °C in mid-diapause (MD) when it significantly increased. Differential scanning calorimetry (DSC) was used to examine phase transitions of total body lipid of cold-acclimated larvae in diapause. Thermal analysis indicated that changes in the melt transition temperatures of whole body total lipids were subtle, but consistent with the modest changes in the level of FA unsaturation observed. We conclude that lipid rearrangements are a function of the endogenous "diapause program" rather than a direct effect of low temperatures, which proved to have limited impact on lipid changes in diapausing larvae of O. nubilalis.

Metabolomic Analysis of Diapausing and Noni-diapausing Larvae of the European Corn Borer Ostrinia nubilalis (Hbn.) (Lepidoptera: Crambidae).

In this study, an (1)H-NMR -based metabolomic approach was used to investigate the biochemical mechanisms of diapause and cold hardiness in diapausing larvae of the European corn borer Ostrinia nubilalis. Metabolomic patterns in polar hemolymph extracts from non-diapausing and diapausing larvae of O. nubilalis were compared. Analysis indicated 13 metabolites: 7 amino acids, glycerol, acetate, citrate, succinate, lactate and putrescine. Results show that diapausing larvae display different metabolomic patterns compared to active non-diapausing larvae, with predominant metabolites identified as glycerol, proline and alanine. In specific diapausing larvae initially kept at 5 °C then gradually chilled to ­3 °C and ­16 °C, alanine , glycerol and acetate were predominant metabolites. (1)H-NMR spectroscopy provides new insight into the metabolomic patterns associated with cold resistance and diapause in O. nubilalis larvae, suggesting distinct metabolomes function in actively developing and diapausing larvae.

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.

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.

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.

Cold defence responses: the role of oxidative stress.

Low temperatures provoke increased production of heat accompanied by increased respiration, oxygen consumption and the production of partially reduced oxygen species called ROS. ROS induce different forms of cellular oxidative damage, disturb the redox state and can change the activity of several metabolic enzymes. Organisms have developed a functionally connected set of anti-oxidant enzymes and low molecular mass compounds (together termed the ADS) that metabolise primary ROS. If ROS production within cells overwhelms the ADS, oxidative damage arises and oxidative stress can occur. Short-term cold exposure in endotherms leads to oxidative stress. As cold exposure persists organisms develop adaptive changes toward reducing ROS production and increasing the ADS. In contrast, heterotherms and ectotherms as a normal part of their over-wintering strategy slow down metabolism, oxygen consumption and subsequently cause ROS production. Increased baseline activity of key anti-oxidant enzymes as well as 'secondary' enzymatic defence and/or glutathione levels in preparation for a putative oxidative stressful situation arising from tissue re-oxygenation seems to be the preferred evolutionary adaptation of such animals exposed to low environmental temperatures.

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

BACKGROUND: Insects 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. RESULTS: A 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. CONCLUSION: Microarray 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.

Surviving extreme polar winters by desiccation: clues from Arctic springtail (Onychiurus arcticus) EST libraries.

BACKGROUND: Ice, snow and temperatures of -14 degrees 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. RESULTS: 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. CONCLUSION: 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.

Assessing pollution in the Danube River near Novi Sad (Serbia) using several biomarkers in sterlet (Acipenser ruthenus L.).

The response of wild fish to pollutants was studied in sterlet (Acipenser ruthenus L.) collected in 2001 and 2002 at two sampling sites in the Danube River near Novi Sad (Serbia): in the vicinity of the oil refinery and at the Danube-Begec, remote from the oil refinery and considered a reference site. The following biomarkers were measured in sterlet collected from these two sites: the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), and glutathione S-transferase and the induction of CYP1A1 in liver and the activities of aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase in serum. The results demonstrated increase in the activity of SOD and GSH-Px in sterlet collected from the Danube-oil refinery (DOR) compared to that from the reference site, while no differences were found in other enzymes. In conclusion, the overall results suggest that an alteration in the activity of SOD and GSH-Px during the observed period reflects the presence of certain prooxidative compounds that can lead to oxidative stress in the liver of sterlet at the DOR site.

Seasonal variations of cytosolic antioxidant enzyme activities in the liver and white muscle of thinlip gray mullet (Liza ramada Risso) from the Adriatic Sea.

The activity of cytosolic antioxidative defence enzymes in the liver and white muscle of thinlip gray mullet (Liza ramada Risso) were compared in winter and spring in the Adriatic Sea. Activity of antioxidative enzymes is functionally organized due to metabolic demands: analyses of variance and correlation analysis revealed tissue- and seasonal- specific organization of antioxidative enzymes. In winter GST activity increased in both tissues compared with spring. At the same time decreased GSH-Px and GR activities were observed and this effect was more pronounced in liver then in white muscle. From correlation analyses it is concluded that the antioxidative components correlate, but the composition of the antioxidative defence system is different in respect to season and tissue. This means that the antioxidative defence system reorganizes its structure due to oxidative demands and to protect the tissues against reactive oxygen species and to establish homeostasis. Discriminant analyses separated groups according to the complete organization of individual components of the system very well and identified individual components (CAT, GST and GR) which contribute most to the differences. Statistical differences were observed between enzyme activities in tissues (liver and muscle) in both winter and spring, and between seasons (winter and spring) for liver tissue only. Since environmental parameters, such as temperature and oxygen concentration in the sea differ with season, we conclude that in this species the tissues examined expressed their antioxidative defence systems in different ways in respect of external/environmental conditions. We propose that tissue- and seasonal- specific levels of antioxidant enzyme activities should be considered in the interpretation of data from future biomonitoring field studies, especially in relation to low temperature.