Prolonged Heat Stress during Winter Diapause Alters the Expression of Stress-Response Genes in Ostrinia nubilalis (Hbn.).

During diapause, a state of temporarily arrested development, insects require low winter temperatures to suppress their metabolism, conserve energy stores and acquire cold hardiness. A warmer winter could, thus, reduce diapause incidence and duration in many species, prematurely deplete their energy reserves and compromise post-diapause fitness. In this study, we investigated the combined effects of thermal stress and the diapause program on the expression of selected genes involved in antioxidant defense and heat shock response in the European corn borer Ostrinia nubilalis. By using qRT-PCR, it has been shown that response to chronic heat stress is characterized by raised mRNA levels of grx and trx, two important genes of the antioxidant defense system, as well as of hsp70 and, somewhat, of hsp90, two major heat shock response proteins. On the other hand, the expression of hsc70, hsp20.4 and hsp20.1 was discontinuous in the latter part of diapause, or was strongly controlled by the diapause program and refractory to heat stress, as was the case for mtn and fer, genes encoding two metal storage proteins crucial for metal ion homeostasis. This is the first time that the effects of high winter temperatures have been assessed on cold-hardy diapausing larvae and pupae of this important corn pest.

Management of inorganic elements by overwintering physiology of cold hardy larvae of European corn borer (Ostrinia nubilalis, Hbn.).

The European corn borer (Ostrinia nubilalis, Hbn.), enters diapause, a strategy characterized by arrest of development and reproduction, reduction of metabolic rate and the emergence of increased resistance to challenging seasonal conditions as low sub-zero winter temperatures. The aim of this study was to investigate the potential role of inorganic elements in the ecophysiology of O. nubilalis, analysing their content in the whole body, hemolymph and fat body, both metabolically active, non-diapausing and overwintering diapausing larvae by ICP-OES spectrometer following the US EPA method 200.7:2001. O nubilalis as many phytophagous lepidopteran species maintain a very low extracellular sodium concentration and has potassium as dominant cation in hemolymph of their larvae. Changes in hemolymph and the whole body sodium content occur already at the onset of diapause (when the mean environmental temperatures are still high above 0 ºC) and remain stable during the time course of diapause when larvae of this species cope with sub-zero temperatures, it seems that sodium content regulation is rather a part of diapausing program than the direct effect of exposure to low temperatures. Compared to non-diapausing O. nubilalis larvae, potassium levels are much higher in the whole body and fat body of diapausing larvae and substantially increase approaching the end of diapause. The concentration of Ca, Mg, P and S differed in the whole body, hemolymph and fat body between non-diapausing and diapausing larvae without a unique trend during diapause, except an increase in their contents at the end of diapause.

High-Risk Human Papillomavirus in Patients with Oral Carcinoma and Oral Potentially Malignant Disorders in Serbia-A Pilot Study.

Background and Objectives: Oral squamous cell carcinoma (OSCC) accounts for about 95% of oral cancers. It represents a serious public health problem due to the high degree of morbidity and mortality, as well as multifactorial etiology. Human papillomavirus (HPV) infection is a well-documented risk factor for oropharyngeal carcinoma, but its role in oral carcinogenesis is still debatable. Our aim was to investigate the differences in the prevalence of high-risk HPV genotypes (HR-HPV) in patients with OSCC and oral potentially malignant disorders (OPMD) from that of healthy subjects. Materials and Methods: A total of 90 subjects were included in the cross-sectional study and divided into three groups of 30 patients each: (1) patients with OSCC, (2) patients with OPMD, and (3) healthy subjects. We examined the presence of 12 HR-HPV genotypes in the obtained biological material (oral swabs) using real-time PCR. Results: One or more of the 12 tested HR-HPV genotypes were detected in 5/30 patients with OSCC and 2/30 with OPMD, whereas no healthy subjects were positive for any of the tested genotypes. There was a statistically significant difference in nodal involvement between HPV-positive and HPV-negative patients with OSCC. Conclusions: Oral HR-HPV was detected in patients with oral premalignant and malignant lesions but not in healthy individuals, suggesting a possible role in oral carcinogenesis. Broad HR-HPV panel testing could increase the sensitivity of risk assessment and screening for OSCC.

Tools in the Era of Multidrug Resistance in Bacteria: Applications for New Antimicrobial Peptides Discovery.

Antimicrobial peptides (AMPs) are small molecules belonging to innate immunity that act against bacteria, fungi, and viruses. With the spread of bacterial strains resistant to current antibiotics, the scientific community is deeply committed to the identification and study of new molecules with putative antimicrobial activity. In this context, AMPs represent a promising alternative to overcome this problem. To date, several databases have been built up to provide information on the AMPs identified so far and their physico-chemical properties. Moreover, several tools have been developed and are available online that allow to highlight sequences with putative antimicrobial activity and predict their biological activity. These tools can also predict the secondary and tertiary structures of putative AMPs, thus allowing molecular docking studies to evaluate potential interactions with proteins/ligands. In this paper, we focused our attention on online available AMPs databases and computational tools for biological activity and tertiary structure prediction, highlighting some papers in which the computational approach was successfully used. As the identification of peptides starts from the analysis of a large amount of data, we show that bioinformatics predictions are the best starting point for the identification of new sequences of interest that can be subsequently produced and tested.

Identification of Intrinsically Disordered Proteins and Regions in a Non-Model Insect Species Ostrinia nubilalis (Hbn.).

Research in previous decades has shown that intrinsically disordered proteins (IDPs) and regions in proteins (IDRs) are as ubiquitous as highly ordered proteins. Despite this, research on IDPs and IDRs still has many gaps left to fill. Here, we present an approach that combines wet lab methods with bioinformatics tools to identify and analyze intrinsically disordered proteins in a non-model insect species that is cold-hardy. Due to their known resilience to the effects of extreme temperatures, these proteins likely play important roles in this insect's adaptive mechanisms to sub-zero temperatures. The approach involves IDP enrichment by sample heating and double-digestion of proteins, followed by peptide and protein identification. Next, proteins are bioinformatically analyzed for disorder content, presence of long disordered regions, amino acid composition, and processes they are involved in. Finally, IDP detection is validated with an in-house 2D PAGE. In total, 608 unique proteins were identified, with 39 being mostly disordered, 100 partially disordered, 95 nearly ordered, and 374 ordered. One-third contain at least one long disordered segment. Functional information was available for only 90 proteins with intrinsic disorders out of 312 characterized proteins. Around half of the 90 proteins are cytoskeletal elements or involved in translational processes.

Acclimations to Cold and Warm Conditions Differently Affect the Energy Metabolism of Diapausing Larvae of the European Corn Borer Ostrinia nubilalis (Hbn.).

The European corn borer Ostrinia nubilalis is a pest species, whose fifth instar larvae gradually develop cold hardiness during diapause. The physiological changes underlying diapause progression and cold hardiness development are still insufficiently understood in insects. Here, we follow a complex of changes related to energy metabolism during cold acclimation (5°C) of diapausing larvae and compare this to warm-acclimated (22°C) and non-diapause controls. Capillary electrophoresis of nucleotides and coenzymes has shown that in gradually cold-acclimated groups concentrations of ATP/ADP and, consequently, energy charge slowly decrease during diapause, while the concentration of AMP increases, especially in the first months of diapause. Also, the activity of cytochrome c oxidase (COX), as well as the concentrations of NAD+ and GMP, decline in cold-acclimated groups, until the latter part of diapause, when they recover. Relative expression of NADH dehydrogenase (nd1), coenzyme Q-cytochrome c reductase (uqcr), COX, ATP synthase (atp), ADP/ATP translocase (ant), and prohibitin 2 (phb2) is supressed in cold-acclimated larvae during the first months of diapause and gradually increases toward the termination of diapause. Contrary to this, NADP+ and UMP levels significantly increased in the first few months of diapause, after gradual cold acclimation, which is in connection with the biosynthesis of cryoprotective molecules, as well as regeneration of small antioxidants. Our findings evidence the existence of a cold-induced energy-saving program that facilitates long-term maintenance of larval diapause, as well as gradual development of cold hardiness. In contrast, warm acclimation induced faster depletion of ATP, ADP, UMP, NAD+, and NADP+, as well as higher activity of COX and generally higher expression of all energy-related genes in comparison to cold-acclimated larvae. Moreover, such unusually high metabolic activity, driven by high temperatures, lead to premature mortality in the warm-acclimated group after 2 months of diapause. Thus, our findings strongly support the importance of low temperature exposure in early diapause for gradual cold hardiness acquisition, successful maintenance of the resting state and return to active development. Moreover, they demonstrate potentially adverse effects of global climate changes and subsequent increase in winter temperatures on cold-adapted terrestrial organisms in temperate and subpolar regions.

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.

The influence of low temperature and diapause phase on sugar and polyol content in the European corn borer Ostrinia nubilalis (Hbn.).

The European corn borer (ECB, Ostrinia nubilalis Hbn.) is a major pest in temperate regions of Europe and North America. Fifth instar ECB larvae enter diapause before winter and gradually develop cold hardiness. Here we investigated the combined influence of diapause phase and low temperature on sugar and polyol content in ECB larvae. Larvae in mid-diapause or diapause termination were acclimated at 5 °C, -3°C or -16 °C, and sugar and polyol content was measured using GC-MS. Control GC-MS measurements were conducted on untreated non-diapausing larvae. We detected differences in polyol (glycerol, sorbitol, myo-inositol) and sugar (trehalose, fructose, glucose) levels in diapausing versus non-diapausing larvae. Glycerol and trehalose were the most abundant of all analyzed cryoprotective compounds in diapausing larvae. Exposure of diapausing larvae to decreasing temperatures induced changes in polyol and sugar levels that depended on the phase of diapause. In mid-diapause larvae, decreasing temperatures induced a significant increase in glycerol and a decrease in sorbitol and myo-inositol. In larvae at diapause termination, polyol content was lower and less influenced by decreasing temperatures. In contrast, sugar levels were lower in larvae at mid-diapause versus diapause termination. Exposure of larvae to -16 °C induced a significant increase in the levels of all detected sugars. In particular, glucose levels were significantly higher in larvae at diapause termination following exposure to -16 °C. We propose that this shift toward sugar synthesis following low temperature exposure in larvae at diapause termination is a consequence of NADPH dependent polyol synthesis, and may be a mechanism for preservation of carbon reserves needed for post-diapause development.

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.

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.

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.