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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.

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.

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

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.

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