Exposure to High Concentrations of Cadmium Which Delay Development of Ostrinia Nubilalis Hbn. Larvae Affected the Balance of Bioelements.

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.

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.

Spermidine supplementation in honey bees: Autophagy and epigenetic modifications.

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.

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.

Spermidine dietary supplementation and polyamines level in reference to survival and lifespan of honey bees.

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.

Identification of a metallothionein gene and the role of biological thiols in stress induced by short-term Cd exposure in Ostrinia nubilalis.

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.

The effects of 6-month hydrogen-rich water intake on molecular and phenotypic biomarkers of aging in older adults aged 70 years and over: A randomized controlled pilot trial.

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).

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.

Effect of fullerenol nanoparticles on oxidative stress induced by paraquat in honey bees.

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.

The effect of long term exposure to cadmium on Ostrinia nubilalis growth, development, survival rate and oxidative status.

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.

Laboratory bioassays on the response of honey bee (Apis mellifera L.) glutathione S-transferase and acetylcholinesterase to the oral exposure to copper, cadmium, and lead.

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.

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.

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.

The impact of sublethal concentrations of Cu, Pb and Cd on honey bee redox status, superoxide dismutase and catalase in laboratory conditions.

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.

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.

Diapause induces changes in the composition and biophysical properties of lipids in larvae of the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae).

This study compares the composition and biophysical properties of lipids in non-diapausing and diapausing fifth instar larvae of Ostrinia nubilalis Hubn. (Lepidoptera: Crambidae). The majority of fat body lipids in both of these physiological states were comprised of ~90% triacylglycerols (TAGs), whereas the haemolymph contained a more even distribution of all lipid classes. The fatty acid composition and biophysical properties of the fat body lipids differed markedly between non-diapausing and diapausing larvae. Diapause was associated with a dramatic increase in the proportions of palmitoleic acid (16:1n-7) and oleic acid (18:1n-9), with concurrent reductions in palmitic acid (16:0) and linoleic acid (18:2n-6). The increase in the level of unsaturation of the fat body lipids, which caused a marked shift in their phase transitions to lower temperatures, was triggered by diapause rather than low temperatures. Adjustments of fatty acid compositions are likely to be an important component of winter diapause mechanisms, possibly maintaining the fluidity of cell membranes and the functionality of the organism during lower winter temperatures.