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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.

In maize plants the attack of European corn borer can cause significant damages, which can lessen by insecticide application in crop protection. The aim of this study was to determine the effect of insecticides on the total length of channels in maize plant stem formed by the feeding of larvae Ostrinia nubilalis. The research was carried out in Maize Research Institute different FAO ripening groups (ZP 434, ZP 600 and ZP 666) and three insecticides chlorantraniliprole (200 g l-1), bifenthrin (100 g l-1) and lufenuron (50 g l-1)+(cypermethrin (50 g l-1) + chlorpyrifos (500 g l-1)), which were applied only after the maximum flight of the first generation to protect plants from attack of European corn borer. The result showed significantly different total length of channels in the stem of plants, caused by feeding of the larvae of European corn borer, which varied from the lowest on treatment with insecticide chlorantraniliprole in maize genotype ZP 600 (170.00 cm) to the highest in maize genotype ZP 666 (278.33 cm) on the control variant (without insecticide application). For all treatments, the average value of total length of channels in stem, was the lowest 192.92 cm in ZP 434, slightly higher 195.42 cm in ZP 600, while the highest average value of total length of channels was 233.34 cm in maize genotype ZP 606. The established differences for total length of channels depended on genotype and type of insecticide applied

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.

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 mortal

During last years, the total losses from European corn borer (Ostrinia nubilalis Hbn.) can reach a high threshold of harmfulness although the different agro-technical measures that are being implemented. According to the sensitivity of hybrids, the damage may vary from only a few percent in tolerant hybrids, up to 30-40% on moderately intolerant hybrids. The aim of this study was to evaluate the variability and bioresponsiveness of four maize genotypes (ZP 427, ZP 434, ZP 600 and ZP 666) to applied insecticides and bioinsecticides and the interdependence of population dynamics. The research has been conducted in 2016 and 2018 in accordance with the EPPO method in field conditions in maize crops. Out of a total of 11.023 attacks in both years of research, in 2016. the largest number of plants (4.841) were with a broken stalk above the ear and at the height of the ear and belonged to categories C3 to C8. In 2018. 3.672 plants were dominated by leaf damage and perforation in the stalk type H2. Analyzing the effectiveness of the tested insecticides and bioinsecticides by application times, an uniformity in the number of plants with recorded symptoms of damage was observed. Thus, foliar treatment with chlorantraniliprole insecticide proved to be the most effective with the least number of attacked plants with a high degree of damage. In contrast, bioinsecticide treatment of plants, in addition to a large number of infested plants, was dominated by high categories of damage that deviated from all other treatments as well as from the control. Within the definition of the degree of tolerance of maize hybrids, the influence of treatment on changes in secondary metabolites (phytins) in seeds was observed. It was noticed that there is a trend of positive correlation of the amount of phytin in the seed (mg/g) with the attack of moths of the first generation and the number of dissected larvae, as well as a trend of negative correlation with the percentage of attacks of moths of

Ostrinia nubilalis Hbn. je štetočina kukuruza koja u pojedinim godišnjim dobima može naneti veliku štetu biljkama. Cilj ovog rada je proučavanje otpornosti odnosno podložnosti hibrida napadu kukuruznog plamenca i efekata različitih doza insekticida i vremena primene u zaštiti od ove štetočine na biljkama kukuruza, ali prvenstveno uticaj hibrida na intenzitet napada. Šest hibrida kukuruza različitih grupa zrenja (ZP 427, ZP 434, ZP 555, ZP 600, ZP 606 i ZP 666) korišćeno je za proučavanje dejstva napada Ostrinia nubilalis Hbn., na kontrolnu varijantu T1 bez primene insekticida i na varijantu tretiranu sa dva insekticida Fobos EC (bifentrin - 200 ml ha-1) i kombinacijom Match 050 EC i Nurelle D (lufenuron 50 g l-1 i hlorpirifos 500 g l-1 + cipermetrin 50 g l-1), 0.75 l ha-1) u dva termina (u prvoj generaciji za vreme leta insekata T2 i T3 i u drugoj generaciji za vreme leta insekata T4 i T5) u tri ponavljanja. Rezultati su pokazali najveći intenzitet Ostrinia nubilalis Hbn. napad i stepen oštećenja na kontrolnoj varijanti i značajne razlike između tretmana za ispitivane parametre. Posmatrajući sve hibride i tretmane, nije utvrđena statistička značajnost između šest ispitivanih hibrida kukuruza različitih grupa zrelosti. U kontrolnoj varijanti najveći napad (oštećenje) je bio na biljkama kod hibrida ZP 666 (94,28%) i ZP 606 (93,90%), a među varijantama primenjenih insekticida najveća šteta je konstatovana u tretmanu T5 sa primenjena kombinacija Match + Nurelle u hibridu ZP 555 i iznosila je 92,02%. Na tretmanu T1-kontrolna varijanta bez insekticida, najmanji napad sa Ostrinia nubilalis Hbn. je konstatovan kod hibrida ZP 434 (od 88,76%), a među varijantama primenjenih insekticida najmanji napad je konstatovan kod varijante T2 sa primenjenim Fobos kod prve generacije insekata u hibridu ZP 434 i iznosio je 77,12%. Napad Ostrinia nubilalis Hbn. i varijacija prosečne mase klipa kukuruza varirala je u zavisnosti od hibrida kukuruza i vremena primene insekticida., Ostrinia nubilalis Hbn. is a corn pest that can do great damage to plants in some seasons. The aim of this paper is to study the resistance / susceptibility of hybrids to the attack of European corn borer and the effects of different doses of insecticides and time of application in protection against pests on maize plants, but primarily the influence of hybrids on attack intensity. The six maize hybrids of different maturation groups (ZP 427, ZP 434, ZP 555, ZP 600, ZP 606 and ZP 666) used for study effect of attack of Ostrinia nubillalis Hbn., on control variant T1 without application of insecticide and on variant treated with two insecticides Fobos EC (bifentrin - 200 ml ha-1 ) and combination of Match 050 EC and Nurelle D (lufenuron 50 g l -1 and hlorpirifos 500 g l -1 + cipermetrin 50 g l -1 ), 0.75 l ha-1 ) in two terms (in the first generation of insect flying T2 and T3 and in the second generation of insect flying T4 and T5) in three replication. The results showed the highest intensity of Ostrinia nubilalis Hbn. attack and degree of damages on control variant and significant differences between treatments for investigated parameters. Observing all hybrids and treatments, there was no statistical significance between the six examined maize hybrids of different maturity groups. In the control variant, the greatest attack (damage) was in plants, namely in the hybrid ZP 666 (94.28%) and ZP 606 (93.90%) and among the variants of applied insecticides, the greatest damage was found in the treatment T5 with the applied combination of Match + Nurelle in hybrid ZP 555 and was 92.02%. On treatment T1-control variant without insecticides, the smallest attack with Ostrinia nubilalis Hbn. was found in the hybrid ZP 434 (from 88.76%) and among the variants of applied insecticides, the smallest attack was found on variant T2 with applied Phobos in the first generation of insect in hybrid ZP 434 and amounted to 77.12%. The attack of Ostrinia nubilalis Hbn. and variation of a

Many insect species overcome unfavourable environmental conditions by entering diapause, a type of hypometabolic resting state which features accumulation of energy reserves, lowered oxidative metabolism rates, synthesis of protective metabolites and expression of specific genes. In order to better understand the changes occuring at the transcriptional level during diapause of the economically important European corn borer Ostrinia nubilalis (Hbn.), the expression of genes encoding selected metabolic enzymes was measured in this study. The selected enzymes are markers of aerobic (citrate synthase, aspartate aminotransferase, alanine aminotransferase) and anaerobic metabolism (lactate dehydrogenase). Total RNA was isolated from whole-body non-diapausing (ND) and diapausing (D) 5th instar larvae acclimated to different temperatures (15°C, 5°C, –3°C, – 16°C) due to a close link between diapause and cold hardiness in this species. QPCR was performed and relative gene expression was determined using rps3 as the reference gene and the ND group as control. Relative expression of cs gene did not differ between groups, suggesting that this enzyme is regulated posttranslationally. Relative expressions of ast and alt genes were significantly higher in the ND group compared to diapausing larvae, while the expression of ldh was higher in the diapausing groups. A temperature effect on relative gene expression of cs, ldh and alt was determined. Higher cs expression was recorded in diapausing larvae acclimated to 5°C compared to D(15°C), probably compensating for lowered CS enzyme activity. Cold acclimation leads to higher expression of ldh in diapausing larvae, especially in D(–3°C) group, as expected. Lastly, acclimating diapausing larvae to 5°C and –3°C resulted in higher expression of alt in these groups compared to D(15°C) larvae, leading to an increase of the cryoprotective alanine synthesis.

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.

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.

Dijapauza je period zaustavljenog razvoja kod insekata, drugih zglavkara i određenih vrsta riba, a koja se javlja na određenom, za svaku vrstu karakterističnom, razvojnom stadijumu. Dijapauzu, kao dugotrajnu i „skupu“ fazu mirovanja, karakteriše prilagođavanje organizma na molekularnom, fiziološkom, morfološkom i bihevioralnom nivou. Promene koje karakterišu dijapauzu većine vrsta su supresija metabolizma, zaustavljanje ili usporavanje razvića, izmenjena ekspresija gena, sinteza zaštitnih proteina i metabolita koji vode ka povećanju otpornosti organizma. Sposobnost insekata da prežive dugotrajne nepovoljne periode u stanju dijapauze omogućila im je da koriste sezonski uslovljene resurse, kolonizuju raznovrsna staništa, smanjuju ukrštanje u srodstvu i povećavaju genetičku raznovrsnost. S obzirom na ogroman značaj dijapauze u životnom ciklusu insekata, ali i drugih organizama, bilo kakvo odlaganje ili njeno prerano prekidanje može imati negativne posledice na njihovo preživljavanje. Iz tog razloga, bolje poznavanje biološke osnove dijapauze može omogućiti unapređenje kontrole insekatskih vrsta koje nanose ekonomsku štetu u poljoprivredi, ali i poboljšanje metoda gajenja vrsta koje su korisne (bumbari, pčele, svilena buba, bubamare, parazitske osice i druge). Takođe, razumevanje mehanizama otpornosti i zaštite od stresa, npr. na niske temperature, mogu doprineti razvoju novih tehnika za krioprezervaciju organa i tkiva. Na kraju, insekti koji miruju, zbog specifičnosti metaboloma, mogu biti potencijalan izvor novih lekova, ali i dobri modeli za istraživanje starenja, gojaznosti i dijabetesa. Kukuruzni plamenac Ostrinia nubilalis (Hübner 1796) je vrsta moljca čije su larve polifagne herbivore koje se hrane na preko 200 biljnih vrsta, uključujući važne poljoprivredne useve, povrće i voće. Krajem leta i početkom jeseni larve 5. stupnja ulaze u fakultativnu dijapauzu, preživljavajući oštre kontinentalne zime u fazi mirovanja. Tokom dijapauze gusenice O. nubilalis razvijaju, Diapause is a carefully regulated state of arrested development that occurs at a species-specific life stage of insects, other arthropods and some fish species. It engages a plethora of molecular, physiological, morphological and behavioral changes that altogether increase stress tolerance and the ability to survive. Thus, diapause enables insects to exploit seasonally variable food resources and colonize new habitats, as well as decrease inbreeding and increase mating of temporally separated genotypes in a population. Due to the tremendous importance of diapause for survival, any factors that delay or perturb diapause can potentially compromise the fitness of an organism. Thus, deeper knowledge of the biology of diapause could help improve or develop new means of pest control for insect populations, as well as to advance current rearing techniques of beneficial species (bees, ladybirds, parasitic wasps, silkworm etc.). Additionally, defining the molecular background of stress tolerance during diapause, such as cold hardiness, could improve techniques for cryopreservation of organs, tissues and cells. Also, because of the specificity of the metabolom, diapausing insects could be regarded as a potentially rich source of new pharmaceutical agents, or could be used as model organisms to study ageing, obesity and diabetes. The European corn borer (ECB), Ostrinia nubilalis (Hübner 1796), is a pest moth species whose polyphagous larvae feed on more than 200 plants, including important crops, vegetables and fruits. In late August or the beginning of September, because of a critical shortening of the photoperiod, 5th instar larvae of ECB stop feeding and enter into diapause. Diapausing larvae gradually develop freeze tolerance and become cold hardy, which has enabled this species to successfully colonize temperate regions. The aim of this dissertation was to improve our understanding of the molecular and biochemical mechanisms governing diapause of O. nubilalis...

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.

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. Conclu