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The honey bee (Apis mellifera L.), as an eusocial insect species, is an important model organism in research focusing on ageing and longevity, due to prominent seasonal lifespan plasticity within the worker caste (summer and winter worker bees). In this study, we employed a screening approach to evaluate several molecular parameters, providing comprehensive insights into the antioxidative (superoxide dismutase and catalase activity, reduced glutathione and sulfhydryl group content, total antioxidative capacity), detoxifying (glutathione S-transferase and acetylcholinesterase activity), and immune (phenol oxidase and glucose oxidase activity) status, as well as vitellogenin content, in the summer and winter generation of honey bees, across ageing stages and in two body compartments: the whole abdomen and the head. Summer worker bees were collected weekly for six weeks, while winter bees were collected monthly for five months. The results of our study clearly indicate a reduced overall antioxidative capacity of older groups of worker bees from both generations, while the parameters of immune responsiveness mostly contributed to the separation between the two generations based on season rather than age categories. Detoxification ability appeared to be more susceptible to environmental factors. An age-dependent increase in vitellogenin content was recorded in the abdomen, but without seasonal differences. These findings provide an excellent starting point for further investigations into age-related changes, particularly within the context of honey bee sociality.

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Polyamines (PAs), including putrescine (Put), spermidine (Spd), and spermine (Spm), are essential polycations with wide-ranging roles in cellular functions. PA levels decline with age, making exogenous PA supplementation, particularly Spd, an intriguing prospect. Previous research in honey bees demonstrated that millimolar Spd added to their diet increased lifespan and reinforced oxidative resilience. The present study is aimed to assess the anti-aging effects of spermidine supplementation at concentrations of 0.1 and 1 mM in honey bees, focusing on autophagy and associated epigenetic changes. Results showed a more pronounced effect at the lower Spd concentration, primarily in the abdomen. Spd induced site-specific histone 3 hypoacetylation at sites K18 and 27, hyperacetylation at K9, with no change at K14 in the entire body. Additionally, autophagy-related genes (ATG3, 5, 9, 13) and genes associated with epigenetic changes (HDAC1, HDAC3, SIRT1, KAT2A, KAT6B, P300, DNMT1A, DNMT1B) were upregulated in the abdomens of honey bees. In conclusion, our findings highlight profound epigenetic changes and autophagy promotion due to spermidine supplementation, contributing to increased honey bee longevity. Further research is needed to fully understand the precise mechanisms and the interplay between epigenetic alterations and autophagy in honey bees, underscoring the significance of autophagy as a geroprotective mechanism.

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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.

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Honey bee health has been an important and ongoing topic in recent years. Honey bee is also an important model organism for aging studies. Polyamines, putrescine, spermidine and spermine, are ubiquitous polycations, involved in a wide range of cellular processes such as cell growth, gene regulation, immunity, and regulation of lifespan. Spermidine, named longevity elixir, has been most analysed in the context of aging. One of the several proposed mechanisms behind spermidine actions is antioxidative activity. In present study we showed that dietary spermidine supplementation: (a) improved survival, (b) increased the average lifespan, (c) influenced the content of endogenous polyamines by increasing the level of putrescine and spermidine and decreasing the level of spermine, (d) reduced oxidative stress (MDA level), (e) increased the antioxidant capacity of the organism (FRAP), (f) increased relative gene expression of five genes involved in polyamine metabolism, and (g) upregulated vitellogenin gene in honey bees. To our knowledge, this is the first study on honey bee polyamine levels in reference to their longevity. These results provide important information on possible strategies for improving honey bee health by introducing spermidine into their diet. Here, we offer spermidine concentrations that could be considered for that purpose.

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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.

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Cadmium (Cd) is a non-essential metal that is highly toxic to all living forms, characterized by an extremely high affinity for thiol (SH) groups. The aim of this work was to identify and experimentally verify metallothionein gene and to analyze the role of biological thiols in stress induced by short-term Cd exposure in Ostrinia nubilalis, one of the most important corn pests. The coding region of a metallothionein (MT) gene in O. nubilalis was identified, encoding protein, OnMT1, which contains 46 amino acids, including 12 cysteine residues, and has no aromatic amino acids. Phylogenetic analysis revealed that OnMT1 clustered together with metallothionein from Bombyx mori. Structural bioinformatics analysis strongly suggests that OnMT1 is a metallothionein with affinity for multiple transition metals. Further, in order to elucidate the role of biological thiols, O. nubilalis L5 larvae were exposed to increasing Cd concentrations in diet (6.85, 41.71, 77.35 mg kg-1) during a 48 h period, after which Cd concentration in larvae was measured (3.50, 12.02, 47.37 mg kg-1, respectively). Due to short-term Cd exposure, concentration of free protein SH groups and relative expression of OnMT1 and thioredoxin (Trx) genes was elevated, while the reduced glutathione content remained unchanged. The presented results provide evidence that OnMT1 plays a role in Cd detoxification and homeostasis, and confirm the importance of biological thiols, especially OnMT1 and Trx, in the early response of O. nubilalis to Cd poisoning, indicating interaction between Cd and thiol-linked redox reactions. Insects provide valuable insight into molecular adaptations to metals.

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Honeybee colony losses have been a focus of research in the last years, due to the importance of managed honeybee colonies for economy and ecology. Different unfavorable conditions from the outside environment have a strong impact on the hive health. The majority of losses occur mainly during winter and the exact reason is not completely understood. Only a small number of studies are dealing with content of bioelements, their function and influence on honeybee physiology. The aim of the present study was to determine seasonal and spatial variations in content of bioelements and non-essential elements, in hemolymph and whole body of honeybees originating from three regions with different degrees of urbanization and industrialization. Concentrations of 16 elements were compared: macroelements (Ca, K, Mg, Na), microelements (Cu, Fe, Mn, Zn) and non-essential elements (Al, Ba, Cd, Co, Cr, Ni, Pb, Sr) in samples collected from 3 different environments: Golija (rural region), Belgrade (urban region) and Zajaca (industrial region). Content of bioelements and non-essential elements in honeybees was under noticeable influence of the surrounding environment, season and degree of honeybee activity. Hemolymph was proven to be helpful in differentiating air pollution from other sources of honeybee exposure. The results of our study demonstrated that bees can be successfully used as biomonitors since we have observed statistically significant differences among observed locations, but unless compared locations are exposed to excessively different pollution pressures, it is essential that all bees should be collected at the same season.

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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.

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In this study the effect of long term exposure to cadmium (Cd) on Ostrinia nubilalis larval growth, development, survival rate and oxidative status was analyzed. Newly hatched first instar - L1 larvae were reared on a Cd contaminated diet until the larvae reached the final, fifth instar - L5 or developed into pupae. In total, six experimental groups, five treatments (concentrations of Cd in fresh diet: Cd I: 0.73, Cd II: 3.70, Cd III: 6.85, Cd IV: 41.71 and Cd V: 77.53 mg kg-1) and a control group (C) were set up. The results of the experiment showed that exposure to higher concentrations of Cd (41.71 and 77.53 mg kg-1) had a significant influence on development and redox status of O. nubilalis larvae: (1) the development rate was strongly reduced resulting in a prolonged pupation time; (2) the survival rate of larvae was prominently lower; (3) bioaccumulation factor (measured in pupae) was reduced which indicated that larvae could accumulate Cd to a certain level; (4) the level of the lipid peroxidation was significantly higher, which points to oxidative damage; (5) the expression of Mtn was significantly up-regulated while Cat and GPx genes down-regulated. In conclusion, long term exposure to dietary Cd in a concentration of 41.7 mg kg-1 and higher, induced oxidative stress and slowed down growth and development of O. nubilalis larvae.

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The enormous progress in nanomaterials development and their use, followed by their inevitable environmental print, has arisen the emerging questions concerning their influence to the living systems. Honey bees are considered to be quite a suitable model system for the risk assessment and prediction of various external influences. To the best of our knowledge, this is the first study dealing with the influence of fullerenol nanoparticles (FNP), a biodegradable carbon nanomaterials' representative, to honey bees. This investigation was conducted with an aim to merge two different open-ended questions: the potential toxic effect of FNP to the bees on the one hand and antioxidative effect of FNP on the other hand. Since FNP antioxidative properties were proved in a number of in vivo models, we hypothesized the similar outcomes, and according to this assumption, we opted for paraquat as a well-known oxidative stress inducer. FNP did not have toxic effect in none of investigated concentrations. The results also confirmed the potential of FNP to reduce oxidative stress through the gene expression of antioxidative enzymes and the change in the redox state of the cells. Additional experiments are needed for a better understanding of the exact mechanism and complex patterns of FNP's activity.

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