Concentration- and Time-Dependent Dietary Exposure to Graphene Oxide and Silver Nanoparticles: Effects on Food Consumption and Assimilation, Digestive Enzyme Activities, and Body Mass in Acheta domesticus.

The advancement of nanotechnology poses a real risk of insect exposure to nanoparticles (NPs) that can enter the digestive system through contaminated food or nanopesticides. This study examines whether the exposure of model insect species-Acheta domesticus-to increasing graphene oxide (GO) and silver nanoparticle (AgNP) concentrations (2, 20, and 200 ppm and 4, 40, and 400 ppm, respectively) could change its digestive functions: enzymes' activities, food consumption, and assimilation. We noticed more pronounced alterations following exposure to AgNPs than to GO. They included increased activity of α-amylase, α-glucosidase, and lipase but inhibited protease activity. Prolonged exposure to higher concentrations of AgNPs resulted in a significantly decreased food consumption and changed assimilation compared with the control in adult crickets. A increase in body weight was observed in the insects from the Ag4 group and a decrease in body weight or no effects were observed in crickets from the Ag40 and Ag400 groups (i.e., 4, 40, or 400 ppm of AgNPs, respectively), suggesting that even a moderate disturbance in nutrient and energy availability may affect the body weight of an organism and its overall condition. This study underscores the intricate interplay between NPs and digestive enzymes, emphasizing the need for further investigation to comprehend the underlying mechanisms and consequences of these interactions.

Does Selection for Longevity in Acheta domesticus Involve Sirtuin Activity Modulation and Differential Response to Activators (Resveratrol and Nanodiamonds)?

Sirtuins, often called "longevity enzymes", are pivotal in genome protection and DNA repair processes, offering insights into aging and longevity. This study delves into the potential impact of resveratrol (RV) and nanodiamonds (NDs) on sirtuin activity, focusing on two strains of house crickets (Acheta domesticus): the wild-type and long-lived strains. The general sirtuin activity was measured using colorimetric assays, while fluorescence assays assessed SIRT1 activity. Additionally, a DNA damage test and a Kaplan-Meier survival analysis were carried out. Experimental groups were fed diets containing either NDs or RV. Notably, the long-lived strain exhibited significantly higher sirtuin activity compared to the wild-type strain. Interestingly, this heightened sirtuin activity persisted even after exposure to RVs and NDs. These findings indicate that RV and NDs can potentially enhance sirtuin activity in house crickets, with a notable impact on the long-lived strain. This research sheds light on the intriguing potential of RV and NDs as sirtuin activators in house crickets. It might be a milestone for future investigations into sirtuin activity and its potential implications for longevity within the same species, laying the groundwork for broader applications in aging and lifespan extension research.

Survival, growth and digestive functions after exposure to nanodiamonds - Transgenerational effects beyond contact time in house cricket strains.

The long-term exposure effects of nanodiamonds (NDs), spanning an organism's entire lifespan and continuing for subsequent generation, remain understudied. Most research has focused on evaluating their biological impacts on cell lines and selected organisms, typically over short exposure durations lasting hours or days. The study aimed to assess growth, mortality, and digestive functions in wild (H) and long-lived (D) strains of Acheta domesticus (Insecta: Orthoptera) after two-generational exposure to NDs in concentrations of 0.2 or 2 mg kg-1 of food, followed by their elimination in the third generation. NDs induced subtle stimulating effect that depended on the strain and generation. In the first generation, more such responses occurred in the H than in the D strain. In the first generation of H strain insects, contact with NDs increased survival, stimulated the growth of young larvae, and the activity of most digestive enzymes in mature adults. The same doses and exposure time did not cause similar effects in the D strain. In the first generation of D strain insects, survival and growth were unaffected by NDs, whereas, in the second generation, significant stimulation of those parameters was visible. Selection towards longevity appears to support higher resistance of the insects to exposure to additional stressor, at least in the first generation. The cessation of ND exposure in the third generation caused potentially harmful changes, which included, e.g., decreased survival probability in H strain insects, slowed growth of both strains, as well as changes in heterochromatin density and distribution in nuclei of the gut cells in both strains. Such a reaction may suggest the involvement of epigenetic inheritance mechanisms, which may become inadequate after the stress factor is removed.

Multigenerational Effects of Graphene Oxide Nanoparticles on Acheta domesticus DNA Stability.

The use of nanoparticles like graphene oxide (GO) in nanocomposite industries is growing very fast. There is a strong concern that GO can enter the environment and become nanopollutatnt. Environmental pollutants' exposure usually relates to low concentrations but may last for a long time and impact following generations. Attention should be paid to the effects of nanoparticles, especially on the DNA stability passed on to the offspring. We investigated the multigenerational effects on two strains (wild and long-lived) of house cricket intoxicated with low GO concentrations over five generations, followed by one recovery generation. Our investigation focused on oxidative stress parameters, specifically AP sites (apurinic/apyrimidinic sites) and 8-OHdG (8-hydroxy-2'-deoxyguanosine), and examined the global DNA methylation pattern. Five intoxicated generations were able to overcome the oxidative stress, showing that relatively low doses of GO have a moderate effect on the house cricket (8-OHdG and AP sites). The last recovery generation that experienced a transition from contaminated to uncontaminated food presented greater DNA damage. The pattern of DNA methylation was comparable in every generation, suggesting that other epigenetic mechanisms might be involved.

Does age pay off? Effects of three-generational experiments of nanodiamond exposure and withdrawal in wild and longevity-selected model animals.

Nanodiamonds (NDs) are considered a material with low toxicity. However, no studies describe the effects of ND withdrawal after multigenerational exposure. The aim was to evaluate ND exposure (in the 1st and 2nd generations) effects at low concentrations (0.2 or 2 mg kg-1) and withdrawal (in the 3rd generation) in the wild (H) and longevity-selected (D) model insect Acheta domesticus. We measured selected oxidative stress parameters, immunity, types of cell death, and DNA damage. Most of the results obtained in the 1st generation, e.g., catalase (CAT), total antioxidant capacity (TAC), heat shock proteins (HSP70), defensins, or apoptosis level, confirmed no significant toxicity of low doses of NDs. Interestingly, strain-specific differences were observed. D-strain crickets reduced autophagy, the number of ROS+ cells, and DNA damage. The effect can be a symptom of mobilization of the organism and stimulation of physiological defense mechanisms in long-living organisms. The 2nd-generation D-strain insects fed ND-spiked food at higher concentrations manifested a reduction in CAT, TAC, early apoptosis, and DNA damage, together with an increase in HSP70 and defensins. ROS+ cells and cells with reduced membrane potential and autophagy did not differ significantly from the control. H-strain insects revealed a higher number of ROS+ cells and cells with reduced membrane potential, decreased CAT activity, and early apoptosis. Elimination of NDs from the diet in the 3rd generation did not cause full recovery of the measured parameters. We noticed an increase in the concentration of HSP70 and defensins (H-strain) and a decrease in apoptosis (D-strain). However, the most visible increase was a significant increase in DNA damage, especially in H-strain individuals. The results suggest prolonged adverse effects of NDs on cellular functions, reaching beyond "contact time" with these particles. Unintentional and/or uncontrolled ND pollution of the environment poses a new challenge for all organisms inhabiting it, particularly during multigenerational exposure.

Age- and Lifespan-Dependent Differences in GO Caused DNA Damage in Acheta domesticus.

The rising applicability of graphene oxide (GO) should be preceded by detailed tests confirming its safety and lack of toxicity. Sensitivity to GO of immature, or with different survival strategy, individuals has not been studied so far. Therefore, in the present research, we focused on the GO genotoxic effects, examining selected parameters of DNA damage (total DNA damage, double-strand breaks-DSB, 8-hydroxy-2'-deoxyguanosine-8-OHdG, abasic site-AP sites), DNA damage response parameters, and global methylation in the model organism Acheta domesticus. Special attention was paid to various life stages and lifespans, using wild (H), and selected for longevity (D) strains. DNA damage was significantly affected by stage and/or strain and GO exposure. Larvae and young imago were generally more sensitive than adults, revealing more severe DNA damage. Especially in the earlier life stages, the D strain reacted more intensely/inversely than the H strain. In contrast, DNA damage response parameters were not significantly related to stage and/or strain and GO exposure. Stage-dependent DNA damage, especially DSB and 8-OHdG, with the simultaneous lack or subtle activation of DNA damage response parameters, may result from the general life strategy of insects. Predominantly fast-living and fast-breeding organisms can minimize energy-demanding repair mechanisms.

Multigenerational selection towards longevity changes the protective role of vitamin C against graphene oxide-induced oxidative stress in house crickets.

This research was designed to investigate changes that can arise in an invertebrate organism due to stress caused by a strong prooxidant, graphene oxide (GO), and a potent antioxidant, vitamin C. The study aimed to investigate if vitamin C may support convalescence after chronic GO intoxication. We investigated the toxicity of chronic dietary graphene oxide administration in house cricket (Acheta domesticus) types: wild and selected for longevity (with a better developed antioxidant system, conducive to long life). Vitamin C was applied immediately after cessation of graphene oxide intoxication to check if it can support the remedial effect. The condition of cells, DNA stability, catalase activity, and the reproduction potential, measured as the Vitellogenin (Vg) protein expression level, were investigated in control and GO treated groups, recovery groups (-GO), and recovery groups with Vit. C (-GO + Vit.C). In this study vitamin C had no evident remedial effect on the house crickets exposed to graphene oxide. Most probably, the mechanism of vitamin C action, in case of intoxication with nanoparticles, is much more complicated. In the context of the results obtained, it is worth considering whether Vit. C, applied after GO intoxication, causes further disturbance of homeostasis in terms of the cells' redox potential.

Multigenerational graphene oxide intoxication results in reproduction disorders at the molecular level of vitellogenin protein expression in Acheta domesticus.

The anthropogenic activities may lead to accumulation of graphene oxide (GO) pollution in the environment. Organisms exposed to chronic or multigenerational GO intoxication can present reproduction depletion. Vitellogenin (Vg) has been used as a parameter for evaluating female fertility due to its importance in embryo nutrition. In this study, we used a promising model organism, Acheta domesticus, which was intoxicated with GO in food for three generations. The aim of the study was to investigate the process of Vg synthesis in crickets depending on the exposure time, GO concentration, and age of the females. The results revealed that chronic GO intoxication had adverse effects on the Vg expression pattern. The 1st generation of insects showing low Vg expression was most affected. The 2nd generation of A. domesticus presented a high Vg expression. The last investigated generation seemed to cope with stress caused by GO, and the Vg expression was balanced. We suggest that the epigenetic mechanisms may play a role in the information transfer to the next generations on how to react to the risk factor and keep reproduction at a high rate. We suspect that chronic GO intoxication can disturb the regular formation of the Vg quaternary structure, resulting in consequences for developing an embryo.

The impact of polystyrene consumption by edible insects Tenebrio molitor and Zophobas morio on their nutritional value, cytotoxicity, and oxidative stress parameters.

The objective of this study was to determine of nutritional value, in vitro cytotoxicity, and oxidative stress parameters in cells of selected insect species (Tenebrio molitor and Zophobas morio) after 30 days of Styrofoam consumption. Furthermore, part of our research is also a consumer survey on the willingness to eat insects fed with Styrofoam (EPS 80). Mealworms fed with Styrofoam were determined to have higher protein (48.66 ± 0.92%) and ash content (4.81 ± 0.22%) with reduced fat (24.05 ± 0.55%) and carbohydrate content (2.95 ± 0.15%) than insects with a conventional diet (48.66 ± 0.92, 2.82 ± 0.12, 43.74 ± 0.77, and 4.78 ± 0.18, respectively) while in the case of superworms, no significant difference in nutrient composition was observed. Moreover, Styrofoam has no influence on the health status of gut cells in examined insects. Additionally, in studied concentrations of insects extracts standardized for protein replacement of the traditional insect diet with polystyrene foam did not increase the cytotoxic properties.

Vitellogenin expression, DNA damage, health status of cells and catalase activity in Acheta domesticus selected according to their longevity after graphene oxide treatment.

The increased use of graphene oxide (GO) raises worrisome questions regarding its possible threat to various ecosystems. Invertebrates represent valuable organisms for environmental studies. The lifespan can influence the ability to cope with toxins, especially those that act via oxidative stress. Two strains of Acheta domesticus, which are selected for longevity, were tested. The main aim was to investigate how GO, when administrated in food, affects: the condition of cells, DNA stability, ROS generation and the reproduction potential (the Vitellogenin (Vg) protein expression). The "recovery effect" - after removing GO from the diet for 15 days - was also measured. The results revealed different responses to GO in the wild (H) and long-living (D) strains. The D strain had a higher catalase activity compared to the H strain on the 25th day of the imago stage. Removing GO from the food resulted in a decrease in the catalase activity to the level of the control. On the 5th day of the imago stage, the H strain had a higher cell mortality than the D strain in the GO-intoxicated groups. There was more DNA damage in the H strain compared to the long-living strain. A remedial effect was seen after the GO was removed from the diet. The total Vg protein expression was higher in the H strain and lower in the D strain. The results indicated a GO concentration-dependent outcome. In both strains, removing the GO from the food led to a high Vg expression. The Vg expression after GO treatment, particularly translation and post-translational processing, should be studied in detail in the future. The D strain of crickets had more specialized mechanisms for maintaining homeostasis than the H strain. Organisms can fight off negative effects of GO, especially when they have systems that are well developed against oxidative stress.

Graphene oxide as a new anthropogenic stress factor - multigenerational study at the molecular, cellular, individual and population level of Acheta domesticus.

Although interest in transgenerational phenomena is constantly growing, little is known about the long-term toxicity of nanoparticles. In this study we investigate the multigenerational effects of graphene oxide (GO) which was given to Acheta domesticus in low doses (0.2, 2 and 20 µg·g-1 of food) for three subsequent generations. We assessed the influence of GO nanoparticles in many contexts, basing on parameters which represented different levels of biological organization: activity of antioxidant enzymes, level of apoptosis, DNA damage, histological analysis, hatching abilities, body mass and body length of insects, as well as their survival rate. The results have shown that exposing insects to nanoparticles over an extended period of time causes surprising intergenerational effects, based on significant differences in the life cycle and reproductive processes, which are not always dose-dependent. The second generation of insects appeared as the most unstable among the parameters that were studied, and did not match trends and patterns in the first and third generation categories. An increase of DNA damage was observed, but only in the third generation. This reduction of genome stability can be perceived as an essential element of adaptation, leading to an increase of genotype variants, which then undergo selection.

Protective role of zinc in Spodoptera exigua larvae under 135-generational cadmium exposure.

The aim of this study was to investigate whether zinc supplementation modulates cadmium toxicity in the beet armyworm Spodoptera exigua selected for 135 generations towards cadmium tolerance. To achieve this, larvae originating from three laboratory populations of S. exigua (control strain - C; cadmium-intoxicated for 135 generations strain - Cd, and control strain intoxicated with Cd for 1 generation - CCd) were additionally exposed to zinc in three concentrations (Zn1, 400 µg Zn·g-1 dry mass of food; Zn2; 200 µg Zn·g-1 dry mass of food; Zn3, 100 µg Zn·g-1 dry mass of food). As the markers of toxicity, a life history traits (the duration of L4 and L5 stages), cellular (DNA damage indices) and biochemical parameters (ADP/ATP ratio and ATP and HSP70 concentrations) were chosen. The duration of larval stages of Zn supplemented larvae was prolonged, while cellular and biochemical indicators, in general, appeared to be lower in comparison to the insects from respective reference groups in each laboratory populations. Moreover, the range of the differences depended on zinc concentration in food. We can suspect that zinc supplementation contributed to the protection of S. exigua individuals against negative effects of cadmium intoxication, probably at the cost of growth rate. Significant differences in the response pattern between insects from different laboratory populations indicate that the influence of additional stress factors is dependent on the overall condition of animals and their previous adaptation to other stressors.

Reduced fecundity and cellular changes in Acheta domesticus after multigenerational exposure to graphene oxide nanoparticles in food.

Despite the fact that the demand for graphene and its derivatives in commercial applications is still growing, many aspects of its toxicity and biocompatibility are still poorly understood. Graphene oxide, which is released into the environment (air, soil and water) as so-called nanowaste or nanopollution, is able to penetrate living organisms. It is highly probable that, due to its specific nature, it can migrate along food chains thereby causing negative consequences. Our previous studies reported that short-term exposure to graphene oxide may increase the antioxidative defense parameters, level of DNA damage, which results in numerous degenerative changes in the gut and gonads. The presented research focuses on reproductive dysfunction and cellular changes in Acheta domesticus after exposure to GO nanoparticles in food (concentrations of 20 and 200 µg·g-1 of food) throughout their entire life cycle. The results showed that long-term exposure to GO caused a significant decrease in the reproductive capabilities of the animals. Moreover, the next generation of A. domesticus had a lower cell vitality compared to their parental generation. It is possible that graphene oxide can cause multigenerational harmful effects.

Gene expression analysis of common carp (Cyprinus carpio L.) lines during Cyprinid herpesvirus 3 infection yields insights into differential immune responses.

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is the etiological agent of a virulent and lethal disease in common and koi carp. This study aimed to determine the genetic basis underlying the common carp immune response to the CyHV-3 virus. Two common carp lines (R3 and K) were infected with CyHV-3 by immersion. The R3 line presented a 20% higher survival rate compared to the K line and significantly lower viral loads as measured at day 3 post infection (p.i.). Microarray analysis using a common carp slides containing a number of 10,822 60-mer probes, revealed that 581 genes in line K (330 up-regulated, 251 down-regulated) and 107 genes in line R3 (77 up-regulated, 30 down-regulated), showed at least a 2-fold difference in expression at day 3 p.i. compared to day 0. Genes which showed at least a 4-fold difference in expression in both lines were selected as potential markers of a CyHV-3 infection in common carp. Additionally, 76 genes showed at least 2-fold differentially expression between K and R3 lines at day 3 p.i. Significantly higher expression of several immune-related genes including number of those which are involve in pathogen recognition, complement activation, MHC class I-restricted antigen presentation and development of adaptive mucosal immunity was noted in more resistant R3 line. Further real-time PCR based analysis provided evidence for higher activation of CD8(+) T cells in R3 line. This study uncovered wide array of immune-related genes involved into antiviral response of common carp toward CyHV-3. It is also demonstrated that the outcome of this severe disease in large extent could be controlled by genetic factors of the host.