Molecular mechanisms of low temperatures survival in polar insects

Sposobnost insekata da se prilagode različitim ekološkim uslovima je veoma dobro dokumentovana; oni predstavljaju najrasprostranjeniju grupu životinja na planeti, sa vrstama koje naseljavaju različita kopnena i vodena staništa, od tropskih predela do polova. Razumevanje mehanizama koji omogućavaju insektima da prežive ekstremne temperature i zadrže vitalne funkcije tokom dugog perioda dormancije je kao model sistem od interesa za mnoge naučne oblasti. Na osnovu načina na koji preživljavaju temperature ispod 0ºC insekte možemo podeliti u tri grupe: i) insekti koji tolerišu formiranje leda u ekstraćelijskom prostoru,ii) insekti koji ne tolerišu zamrzavanje i moraju da ga izbegnu,  a to  čine superhlađenjem svojih telesnih tečnosti i  iii) insekti koji preživljavaju zahvaljujući gubitku vode kroz permeabilnu kutikulu, što je nazvano krioprotektivna dehidratacija. Zajednička odlika organizama sa različitim mehanizmima adaptacije na niske temperature je setbiohemijskih jedinjenja  čija se fiziološka funkcija razlikuje u zavisnosti da li organizam pripada grupi koja toleriše ili ne toleriše zamrzavanje. To su nukleatori kristalizacije leda, krio/anhidroprotektanti i antifriz proteini. Cilj ovih istraživanja je bio ispitivanje molekularne osnove otpornosti na niske temperature dve vrste polarnih kolembola  Onychiurus arcticus i Cryptopygus antarcticus kombinujući fiziološki, biohemijski i molekularno biološki pristup. Ispitivane vrste izbegavaju zamrzavanje svojih telesnih tečnosti primenjujući različite strategije preživljavanja. Za antarktičku vrstu  C. antarcticus karakteristična je brza promena tačke superhlađenja, kao i njena bimodalna distribucija tokom leta, kada neke jedinke mrznu na  višim temperaturama (manje otporne na hladnoću), a druga na nižim (otpornije na hladnoću). Ova bimodalna distribucija tačke superhlađenja je dobro dokumentovana, ali slabo razjašnjena na molekularnom nivou. Druga, arktička vrsta  O. arcticus  koristi strategiju preživljavanja zimskih temperature koje idu i do -25ºC nazvanu krioprotektivna dehidratacija. Na ovaj način, količina slobodne vode u telu se značajno redukuje, a akumulira se trehaloza koja deluje kao  krio/anhidroprotektant. Iako je krioprotektivna dehidratacija opisana i kod drugih vrsta insekata, molekularni mehanizmi koji se nalaze u osnovi ovog fenomena su veoma slabo razjašnjeni. Za karakterizaciju genoma generisano je 16379 EST sekvenci za  O. arcticus i 1180 za  C. antarcticus. To su ujedno i prvi javno dostupni podaci u bazama podataka o genomima ove dve vrste koji predstavljaju značajnu osnovu za komparativne genomske analize. Činjenica da kod obe analizirane vrste, oko 60% EST sekvenci nije pokazalo statistički značajnu sličnost sa proteinima iz baza podataka ukazuje na specifičan patern genske ekspresije kao adaptivni odgovor ispitivanih vrsta na niske temperature. Sa ciljem da se identifikuju geni uključeni u preživljavanje niskih temperatura konstruisani su mikroereji, za  O. arcticus štampanjem 6912 cDNK u duplikatu, a za  C. antarcticus štampanjem 672 cDNK u duplikatu.. Analizom sekvenci identifikovanih putem homologije sa dostupnm bazama podataka kod C. antarcticusuočen je jasan trend povećane ekspresije gena koji kodiraju strukturne proteine u grupi koja je otporna na hladnoću. Ove strukturne proteine uglavnom  čine kutikularni proteini, što je u skladu sa rezultatima nedavnih istraživanja kod kolembola, da je presvlačenje proces tokom kog se snižava tačka superhlađenja, odnosno da varijacije u tački superhlađenja mogu nastati kao posledica endogenih fizioloških procesa tokom presvlačenja. Kod  O. arcticus analizom EST sekvenci i mikroereja identifikovani su potencijalni geni i biohemijski putevi povezani sa krioprotektivnom dehidratacijom, a istakli bi gene uključene u metabolizam ugljenih hidrata, gene za akvaporine, proteine toplotnog stresa, LEA proteine i enzime antioksidativne zaštite.The ability of insects to adapt to diverse ecological conditions iswell documented; they  are the most diverse fauna on earth, with different species occupying arange of terrestrial and aquatic habitats from the tropics to the poles. Understanding the mechanisms by which insects survive such extreme temperatures and retain viability for longperiods in the dormant state is of great interest to many scientific fields. Insects have evolved three main strategies to survive sub-zero temperatures:  i) freeze tolerance,  ii) freeze avoidance and  iii) cryoprotective dehydration. The main biochemical compounds involved in surviving sub-zero temperatures are same for different strategies but their physiological  role is different. They include: ice nucleating agents (INAs), cryo/ anhydroprotectants, and antifreeze proteins (AFPs).  The aim of this study was to determine molecular adaptations to extreme cold  environments, combining physiology, biochemistry and molecular biology  pproaches, in thePolar Collembola:  Cryptopygus antarcticus and  Onychiurus arcticus. Both species are freeze avoiding but employ different strategies for surviving low temperatures. The Antarctic springtail  C. antarcticusis capable of rapid cold hardening with a bi-modal distribution of super cooling points (SCP) with high (less cold-hardened) and low (more  cold-hardened) groups of animals present even during the growing season in summer. This bimodal distribution has been well documented, but is poorly understood. The Arctic springtail  O. arcticusemploys the strategy known as cryoprotective dehydration to survive winter temperatures as low as   -25ºC. With this technique, the amount ofavailable water in the body  is reduced to almost zero and also there is an accumulation of trehalose, which acts as a cryo/anhydroprotectant. Although cryoprotective dehydration has been described in  other  insects, the molecular mechanisms behind this phenomenon are poorly understood. A total of 16,379 EST clones were generated for O. arcticus and 1180 for C. antarcticus. This represents the first publicly available sequence data for this two species providing useful data for comparative genomic analysis. The fact that around 60% of the clones for both species showed no sequence similarity to annotated genes  in the datasets, suggests a specific pattern of gene expression in these species as adaptation to low temperatures. Two microarrays were constructed to identify genes involved in  surviving low temperatures, one for C. antarcticus by printing 672 clones in duplicate and the other  for O. arcticus by  printing 6912 clones in duplicate. An analysis of those where putative function could be inferred via database homology, in C. antarcticus there was aclear 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 recenttheory that summer SCP variation within Collembola species could be a consequence of moulting, with moulting population having lowered SCPs. In O. arcticus EST and microarrayanalysis revealed clones and biochemical pathways associated with cryoprotective dehydration with a particular  reference to genes involved in carbohydrate metabolism, aquaporin  genes, heat shock  proteins, LEA proteins and antioxidant enzymes

The effect of low-dose spermidine supplementation on polyamine content and antioxidative defence mechanisms in honey bees

Summary. The honey bee, a widespread pollinator, contributes to the conservation of biodiversity. In recent decades, a trend of declining colony numbers has emerged. The unsustainable exploitation of the environment may be the cause of this phenomenon. One protective strategy of organisms is to strengthen their antioxidative capacity. A class of positively charged molecules, polyamines, plays important roles in various cellular processes. They exert a regulatory effect on gene expression, have antioxidative properties, and promote longevity in model organisms. The three main representatives are putrescine, spermidine and spermine. The aim of this study was to determine whether supplementation of bees with low-dose spermidine leads to an increased level of the mentioned polyamines and whether this could strengthen the antioxidative defence system. Two experimental groups were established: C group (control), fed with a 50% (w/v) sucrose solution, and S0.01 group, whose diet was supplemented with 0.01 mM spermidine. The experiment lasted for 10 and 17 days. A significant increase in putrescine, spermidine and spermine content was noted in the supplemented group after 17 days, compared to its control. These results show a positive impact of spermidine supplementation on maintaining polyamine levels throughout aging. FRAP and MDA biochemical assays were used for the assessment of oxidative status. FRAP assay showed increased antioxidative capacity in the S0.01 group. These results are in accordance with the results obtained from the MDA assay, which showed a decreased level of lipid peroxidation in the supplemented group, in comparison to the control. The potential practical outcome of this study could be the use of spermidine in beekeeping practice to promote overall honey bee health

Diapause induces remodelling 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 body 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 FAs composition is 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 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 remodelling 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

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

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

Effect of Cold Acclimation on Selected Metabolic Enzymes During Diapause in The European Corn Borer Ostrinia nubilalis Hbn

The European corn borer, Ostrinia nubilalis Hbn., is a pest Lepidopteran species whose larvae overwinter by entering diapause, gradually becoming cold-hardy. To investigate metabolic changes during cold hardening, activities of four metabolic enzymes – citrate synthase (CS), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in whole-body homogenates of pupae, non-diapausing and diapausing larvae acclimated to 5 °C, −3 °C and −16 °C. The highest CS activity was detected in non-diapausing larvae, reflecting active development, while the highest in vitro LDH activity was recorded in diapausing larvae at temperatures close to 0 °C, evidencing a metabolic switch towards anaerobic metabolism. However, in-gel LDH activity showed that production of pyruvate from lactate is triggered by sub-zero temperatures. The activities of both aminotransferases were highest in non-diapausing larvae. Our findings suggest that during diapause and cold hardening the aminotransferases catalyse production of L-alanine, an important cryoprotectant, and L-aspartate, which is closely tied to both transamination reactions and Krebs cycle. The results of this study indicate that, during diapause, the activity of metabolic enzymes is synchronized with exogenous factors, such as temperatures close to 0 °C. These findings support the notion that diapause is metabolically plastic and vibrant, rather than simply a passive, resting state

Molecular mechanisms of low temperatures survival in polar insects

Sposobnost insekata da se prilagode različitim ekološkim uslovima je veoma dobro dokumentovana; oni predstavljaju najrasprostranjeniju grupu životinja na planeti, sa vrstama koje naseljavaju različita kopnena i vodena staništa, od tropskih predela do polova. Razumevanje mehanizama koji omogućavaju insektima da prežive ekstremne temperature i zadrže vitalne funkcije tokom dugog perioda dormancije je kao model sistem od interesa za mnoge naučne oblasti. Na osnovu načina na koji preživljavaju temperature ispod 0ºC insekte možemo podeliti u tri grupe: i) insekti koji tolerišu formiranje leda u ekstraćelijskom prostoru,ii) insekti koji ne tolerišu zamrzavanje i moraju da ga izbegnu,  a to  čine superhlađenjem svojih telesnih tečnosti i  iii) insekti koji preživljavaju zahvaljujući gubitku vode kroz permeabilnu kutikulu, što je nazvano krioprotektivna dehidratacija. Zajednička odlika organizama sa različitim mehanizmima adaptacije na niske temperature je setbiohemijskih jedinjenja  čija se fiziološka funkcija razlikuje u zavisnosti da li organizam pripada grupi koja toleriše ili ne toleriše zamrzavanje. To su nukleatori kristalizacije leda, krio/anhidroprotektanti i antifriz proteini. Cilj ovih istraživanja je bio ispitivanje molekularne osnove otpornosti na niske temperature dve vrste polarnih kolembola  Onychiurus arcticus i Cryptopygus antarcticus kombinujući fiziološki, biohemijski i molekularno biološki pristup. Ispitivane vrste izbegavaju zamrzavanje svojih telesnih tečnosti primenjujući različite strategije preživljavanja. Za antarktičku vrstu  C. antarcticus karakteristična je brza promena tačke superhlađenja, kao i njena bimodalna distribucija tokom leta, kada neke jedinke mrznu na  višim temperaturama (manje otporne na hladnoću), a druga na nižim (otpornije na hladnoću). Ova bimodalna distribucija tačke superhlađenja je dobro dokumentovana, ali slabo razjašnjena na molekularnom nivou. Druga, arktička vrsta  O. arcticus  koristi strategiju preživljavanja zimskih temperature koje idu i do -25ºC nazvanu krioprotektivna dehidratacija. Na ovaj način, količina slobodne vode u telu se značajno redukuje, a akumulira se trehaloza koja deluje kao  krio/anhidroprotektant. Iako je krioprotektivna dehidratacija opisana i kod drugih vrsta insekata, molekularni mehanizmi koji se nalaze u osnovi ovog fenomena su veoma slabo razjašnjeni. Za karakterizaciju genoma generisano je 16379 EST sekvenci za  O. arcticus i 1180 za  C. antarcticus. To su ujedno i prvi javno dostupni podaci u bazama podataka o genomima ove dve vrste koji predstavljaju značajnu osnovu za komparativne genomske analize. Činjenica da kod obe analizirane vrste, oko 60% EST sekvenci nije pokazalo statistički značajnu sličnost sa proteinima iz baza podataka ukazuje na specifičan patern genske ekspresije kao adaptivni odgovor ispitivanih vrsta na niske temperature. Sa ciljem da se identifikuju geni uključeni u preživljavanje niskih temperatura konstruisani su mikroereji, za  O. arcticus štampanjem 6912 cDNK u duplikatu, a za  C. antarcticus štampanjem 672 cDNK u duplikatu.. Analizom sekvenci identifikovanih putem homologije sa dostupnm bazama podataka kod C. antarcticusuočen je jasan trend povećane ekspresije gena koji kodiraju strukturne proteine u grupi koja je otporna na hladnoću. Ove strukturne proteine uglavnom  čine kutikularni proteini, što je u skladu sa rezultatima nedavnih istraživanja kod kolembola, da je presvlačenje proces tokom kog se snižava tačka superhlađenja, odnosno da varijacije u tački superhlađenja mogu nastati kao posledica endogenih fizioloških procesa tokom presvlačenja. Kod  O. arcticus analizom EST sekvenci i mikroereja identifikovani su potencijalni geni i biohemijski putevi povezani sa krioprotektivnom dehidratacijom, a istakli bi gene uključene u metabolizam ugljenih hidrata, gene za akvaporine, proteine toplotnog stresa, LEA proteine i enzime antioksidativne zaštite.The ability of insects to adapt to diverse ecological conditions iswell documented; they  are the most diverse fauna on earth, with different species occupying arange of terrestrial and aquatic habitats from the tropics to the poles. Understanding the mechanisms by which insects survive such extreme temperatures and retain viability for longperiods in the dormant state is of great interest to many scientific fields. Insects have evolved three main strategies to survive sub-zero temperatures:  i) freeze tolerance,  ii) freeze avoidance and  iii) cryoprotective dehydration. The main biochemical compounds involved in surviving sub-zero temperatures are same for different strategies but their physiological  role is different. They include: ice nucleating agents (INAs), cryo/ anhydroprotectants, and antifreeze proteins (AFPs).  The aim of this study was to determine molecular adaptations to extreme cold  environments, combining physiology, biochemistry and molecular biology  pproaches, in thePolar Collembola:  Cryptopygus antarcticus and  Onychiurus arcticus. Both species are freeze avoiding but employ different strategies for surviving low temperatures. The Antarctic springtail  C. antarcticusis capable of rapid cold hardening with a bi-modal distribution of super cooling points (SCP) with high (less cold-hardened) and low (more  cold-hardened) groups of animals present even during the growing season in summer. This bimodal distribution has been well documented, but is poorly understood. The Arctic springtail  O. arcticusemploys the strategy known as cryoprotective dehydration to survive winter temperatures as low as   -25ºC. With this technique, the amount ofavailable water in the body  is reduced to almost zero and also there is an accumulation of trehalose, which acts as a cryo/anhydroprotectant. Although cryoprotective dehydration has been described in  other  insects, the molecular mechanisms behind this phenomenon are poorly understood. A total of 16,379 EST clones were generated for O. arcticus and 1180 for C. antarcticus. This represents the first publicly available sequence data for this two species providing useful data for comparative genomic analysis. The fact that around 60% of the clones for both species showed no sequence similarity to annotated genes  in the datasets, suggests a specific pattern of gene expression in these species as adaptation to low temperatures. Two microarrays were constructed to identify genes involved in  surviving low temperatures, one for C. antarcticus by printing 672 clones in duplicate and the other  for O. arcticus by  printing 6912 clones in duplicate. An analysis of those where putative function could be inferred via database homology, in C. antarcticus there was aclear 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 recenttheory that summer SCP variation within Collembola species could be a consequence of moulting, with moulting population having lowered SCPs. In O. arcticus EST and microarrayanalysis revealed clones and biochemical pathways associated with cryoprotective dehydration with a particular  reference to genes involved in carbohydrate metabolism, aquaporin  genes, heat shock  proteins, LEA proteins and antioxidant enzymes

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

Background Ice, snow and temperatures of -14°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

Exposure to high concentrations of cadmium which delay development of Ostrinia nubilalis Hbn. larvae affected the balance of bioelements

All processes involved in metal homeostasis must be coordinated to provide sufficient, but not toxic, concentrations of important bioelements, and to minimize detrimental effects of toxic metals. Our previous studies dealing with the exposure of O. nubilalis non-diapausing larvae to dietary Cd demonstrated that exposure to higher concentrations of Cd caused delay in the development of larvae, induced oxidative stress and also induced defense mechanisms against the toxic effects of Cd. The aim of the present study was to evaluate how acute and chronic exposure of O. nubilalis larvae to increased concentrations of dietary Cd affected the balance of important bioelements. The concentration of bioelements was analyzed in larvae (after short-term exposure) and pupae (after long-term exposure). The short-term exposure of final instar larvae (L5) to Cd did not affect significantly the concentration of any of the analyzed bioelements, while the long-term exposure of developing larvae to higher concentrations of Cd caused increase in the concentrations of Ca, Mg and Na in pupae. The bioaccumulation factor, calculated for bioelements after long-term exposure to Cd, was higher for the most bioelements in groups fed with diet containing higher concentrations of Cd, except K which displayed the opposite trend. Pearson correlation coefficient showed positive correlations between Cd and Ca, Mg, Na, Fe, Cu and Zn, while negative correlation was observed between Cd and K. The results indicate that impact on the balance of important bioelements might be one of the mechanisms of cadmium toxicity and certainly raise numerous questions for future research