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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. © 2022, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.

The use of insecticides, especially neonicotinoids (NEOCs), poses a significant threat to honey bees, thus prompting the EU to ban all outdoor uses of clothianidin, imidacloprid, and thiamethoxam. However, due to the persistence of NEOCs, as well as the fact that certain NEOCs are in use in some areas of the world, exposure to them is still an on-going issue. In the present study, we used laboratory acute toxicity test to examine the effects of sublethal concentrations (resulting in consumed doses approximately 50, 20, and 5 times below LD50) of thiacloprid and clothianidin on selected parameters of antioxidative defense (superoxide dismutase, catalase, glutathione S-transferase activities), oxidative status (reduced glutathione, protein thiols, malondialdehyde), neurotoxicity (acetylcholinesterase activity), and immune response (prophenoloxidase and phenoloxidase activities) in honey bees. Results indicated that both neonicotinoids’ sublethal concentrations promote oxidative stress in honey bees and affect their immune defense. NEOCs impair detoxification abilities through reduced glutathione S-transferase activity, as well as disturb immune response due to decreased prophenoloxidase and phenoloxidase activities, which can explain the high toxicity of these substances to honey bees. © 2022, INRAE, DIB and Springer-Verlag France SAS, part of Springer Nature.

The aim of the present study was to analyze the mechanisms of ibogaine action by measuring its ex vivo effects on antioxidant defense in the honey bee (Apis mellifera, L.) midgut. The transcriptional levels of selected genes: Cu/Zn dependent and Mn dependent superoxide dismutases (Sod1 and Sod2, respectively), catalase (Cat) and transcription factor Nrf2 (Nrf2) were determined. The applicability of midgut tissue, which expected to have well developed antioxidant protection system, for this type of analysis was confirmed by testing cell viability and response to paraquat, an effective inducer of oxidative stress, ex vivo. Incubation for 2 h with paraquat (10 µg/mL) induced a significant increase in expression of Sod1 and Cat genes. The results of ibogaine treatment showed that exposure to 5 µg/mL and 10 µg/mL of ibogaine for 2 h induced significant increase in expression of Sod1 gene. On the other hand, ibogaine did not lead to a significant increase of Sod2, Cat and transcription factor Nrf2 genes expression in honey bee midgut ex vivo. Our results confirmed positive effect of ibogaine on the antioxidant protective system and its pro-antioxidant action.

© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. 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.

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.

© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. In the present study, the influence of cadmium, copper, and lead on two enzymes often used as biomarkers in toxicological analysis was investigated. Bees were fed with 1 M sucrose solution containing 10-fold serial dilutions of CuCl2 (1000 mg L−1, 100 mg L−1, and 10 mg L−1), CdCl2 (0.1 mg L−1, 0.01 mg L−1, and 0.001 mg L−1), or PbCl2 (10 mg L−1, 1 mg L−1, and 0.1 mg L−1) during 48 h. Our results showed that the total glutathione S-transferase activity was not changed under the influence of cadmium and lead, and it was decreased with the highest concentration of copper. The level of gene expression of the three analyzed classes of glutathione S-transferase was significantly increased with increasing concentrations of copper and cadmium. Lead did not cause significant changes in glutathione S-transferase activity and gene expression, while it showed biphasic effect on acetylcholinesterase activity: lower concentration of lead, 0.1 mg L−1 inhibited and higher dose, 10 mg L−1 induced acetylcholinesterase activity in honey bees. Furthermore, our results showed a significant decrease of the acetylcholinesterase activity in honey bees treated with 0.001 and 0.01 mg L−1 CdCl2. Our results indicate the influence of cadmium, copper, and lead on GST and AChE in the honey bees. These results form the basis for future research on the impact of metallic trace element pollution on honey bees.

Over the past decades, the number of managed honey bee Apis mellifera L. (Hymenoptera: Apidae) colonies have been decreasing. The majority of losses occur during winter, suggesting that overwintering honey bees are more susceptible to adverse factors. We focused on the oxidative status of overwintering honey bees, particularly at the beginning (November) and end (March) of the wintering period. Colonies from three locations with different anthropogenic influences were selected: Belgrade, an urban zone, Zajača, an industrial zone, and Susek, a rural area. We measured levels of malondialdehyde (MDA), as a marker of lipid peroxidation, as well as the expression and activity of select antioxidative enzymes: superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST). Our results show that enzyme activity and gene expression of antioxidative enzymes are influenced by both sample location and the time of sampling. The majority of analyzed genes had significantly reduced expression, at the end of the overwintering period when higher activities of antioxidative enzymes were also recorded. Among the analyzed parameters, SOD activity and gene expression of microsomal GST isoforms were more affected by local environmental conditions, suggesting the complex role of these enzymes in antioxidative defense and detoxification. The higher MDA levels observed at the end of overwintering for all three locations likely reflects accumulated oxidative damage which could be associated with the aging process, brood rearing and/or the onset flying activity.

In this randomized controlled pilot trial, we investigated the effects of a 6-month intake of hydrogen-rich water (HRW) on several molecular and phenotypic biomarkers of aging in older adults aged 70 years and over. Forty older adults (20 women) were randomly allocated in a parallel-group design to receive 0.5 L per day of HRW (15 ppm of hydrogen) or control drink (0 ppm of hydrogen) during a 6-month intervention period. The biomarkers assessed at baseline and 6-month follow up were molecular markers in the blood (DNA and chromosomes, nutrient sensing, protein, and lipid metabolism, oxidative stress and mitochondria, cell senescence, inflammation), brain metabolism, cognitive functioning, physical function and body composition, resting blood pressure, facial skin features, sleep outcomes, and health-related quality of life. The mean age, weight, and height of study participants were 76.0 ± 5.6 years, 78.2 ± 16.1 kg, height 167.5 ± 11.5 cm, respectively. A significant treatment vs. time interaction was found for telomere length (P = 0.049), with the length increased after HRW intervention (from 0.99 ± 0.15 at baseline to 1.02 ± 0.26 at follow up) and decreased after drinking control water (from 0.92 ± 0.27 to 0.79 ± 0.15). A marker of DNA methylation (Tet methylcytosine dioxygenase 2, TET2) expression at 6-month follow-up increased in both groups, yet the degree of elevation was significantly higher in HRW (from 0.81 ± 0.52 at baseline to 1.62 ± 0.66 at follow up) comparing to the control water (from 1.13 ± 0.82 to 1.76 ± 0.87) (P = 0.040). A strong trend for treatment vs. time interaction was found for a degree of DNA methylation (P = 0.166), with the methylation increased in the HRW group (from 120.6 ± 39.8 ng at baseline to 126.6 ± 33.8 ng at follow up) and decreased after taking control water (from 133.6 ± 52.9 ng to 121.2 ± 38.4 ng). HRW was superior to control water to increase brain choline and NAA levels in the left frontal grey matter, brain creatine at the right parietal white matter, and brain NAA at the right parietal mesial grey matter (P < 0.05). No significant differences were found between interventions for other outcomes (P > 0.05), except for a significantly improved chair stand performance after HRW intervention compared to the control water (P = 0.01). Owing to pleiotropic mechanisms of hydrogen action, this simple biomedical gas could be recognized as a possible anti-aging agent that tackles several hallmarks of aging, including loss of function and telomere length shortening. The study was registered at ClinicalTrials.gov (NCT04430803).

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.

© 2018 Elsevier Ltd 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.

The recent decline in managed honey bee populations, Apis mellifera L. (Hymenoptera: Apidae), has caused scientific, ecological, and economic concern. Research into the formation of reactive oxygen species (ROS), antioxidative defense mechanisms, and oxidative stress can contribute to our understanding of bee survival and conservation of this species. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) enzymes together with levels of malondialdehyde (MDA) were measured in summer and winter honey bees sampled from three colonies. One colony was stationary (C1), entering the winter period having accumulated Robinia pseudoacacia L. (Fabaceae) honey, and two were migratory (C2 and C3), entering the winter period with mainly Tilia (Malvaceae) and Brassica (Brassicaceae) honey, respectively. Compared to summer workers, winter worker bees had decreased SOD and GST activity, and MDA level, whereas CAT activity increased in all three colonies. We also demonstrated that seasonality is the main factor responsible for changes in antioxidant enzymes and MDA levels in worker honey bees. Overall, our results indicate a difference between summer and winter worker bees, pointing at a reduced level of antioxidant enzyme defenses during overwintering which may be due to a decrease in production of ROS. The decreased levels of MDA measured in winter honey bees confirm this. As ROS are actively used by insects as a defense mechanism to fight pathogens, we suggest that reduced production of ROS contributes to higher susceptibility of winter honey bees to infections and reduced overwinter survival.

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.

In this study, laboratory bioassays were performed to investigate the impact of sublethal concentrations of Cu (CuCl2: 1000, 100, 10 mg L(-1)), Pb (PbCl2: 10, 1, 0.1 mg L(-1)) and Cd (CdCl2: 0.1, 0.01, 0.001 mg L(-1)) on honey bee redox status and the activity of the main antioxidative enzymes and their gene expression. Our results show that exposure to these metals led to significant changes of gene expression, the levels of enzyme activity and redox status, but the effects are metal and dose dependent. In general, exposure of 48 h to given concentrations of Cu, Cd and Pb did not change the activity of antioxidative enzymes and the level of lipid peroxidation, with the exception of decreased activity of catalase at the lowest concentration of cadmium. Only lead produced increases in glutathione and thiol groups. Expression of genes for catalase and superoxide dismutase changed with exposure to cadmium and copper, whilst lead induced only expression of superoxide dismutase genes. The results from this study provide basic data for future research regarding the impacts of metal pollution on Apis mellifera and will be an important step towards a comprehensive risk assessment of the environmental stressors on honey bees.

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.

Nuclear magnetic resonance (NMR) spectroscopy is powerful metabolomic technique which allows overview of numerous compounds simultaneously. In this study, 1H-NMR -based metabolomic approach was used to investigate entering into diapause and cold hardiness of diapausing larvae of European corn borer (Ostrinia nubilalis). Obvious relationship between diapause and cold hardiness of this species is poorly understood, especially on metabolomic level. Therefore, we have compared the metabolomic patterns in polar extract of hemolymph of non-diapausing and diapausing larvae of O. nubilalis. Results have shown that diapauses, as dormant state, has different metabolomic pattern compared to active non-diapausing phase. Furthermore, differences were indicated among diapausing larvae that were at 5 °C and chilled to -3 °C and -16 °C for two weeks. This separation includes 13 metabolites: seven amino acids, glycerol, acetate, citrate, succinate, lactate and putrescine. 1H-NMR spectroscopy gave new insight into metabolomics of cold resistance and diapause of O. nubilalis indicating distinct metabolomes of actively developing and diapausing larvae.