Assessing the toxicity of green Agaricus bisporus-based Cadmium Sulfide nanoparticles on Musca domestica as a biological model.

The common housefly, Musca domestica, known for transmitting over 100 infections, was studied using green-synthesized Cadmium Sulfide nanoparticles (CdS NPs) from Agaricus bisporus. These CdS NPs were tested on third-instar larvae under laboratory conditions using dipping and feeding methods with concentrations (75, 100, 125, 150, 175, and 200 µg/mL). The toxicity, measured by LC50, was found to be 138 µg/mL for dipping treatment and 123 µg/mL for feeding treatment. Analysis with an energy-dispersive X-ray microanalyzer confirmed Cd accumulation in the larval midgut, indicating penetration of CdS NPs into the organism, which may potentially increase their toxicity. CdS NPs caused disruptions in Heat Shock Protein 70, cell apoptosis, and various biochemical components. Scanning electron microscopy revealed morphological abnormalities in larvae, pupae, and adults exposed to CdS NPs. Ultrastructural examination showed significant midgut tissue abnormalities in larvae treated with 123 µg/mL of CdS NPs. Our study demonstrated that green-synthesized CdS NPs from A. bisporus can effectively control the development of M. domestica larvae.

Efficacy of propolis and royal jelly in attenuating cadmium-induced spermatogenesis and steroidogenesis dysregulation, causing infertility in rats.

Bee-derived pharmaceutical products, including propolis (PRO) and royal jelly (ROJ), possess outstanding pharmacological properties. However, their efficiency in counteracting the deleterious influences of cadmium (Cd) in testes and the relevant mechanisms entail further investigations. Therefore, this study sheds light on the therapeutic efficacy of PRO and ROJ against testicular dysfunction and infertility induced by Cd. Toward this end, 30 mature male Wistar albino rats were randomly divided into six groups (5 animals/group), including (I) control, (II) Cd, (III) PRO, (IV) ROJ, (V) PRO + Cd, and (VI) ROJ + Cd groups. Furthermore, antioxidant factors, semen quality, hormonal levels, steroidogenic enzymes, and genotoxicity were assessed. Moreover, histopathological and ultrastructural attributes and offspring rates were investigated. The Cd-treated group revealed marked reductions in reduced glutathione (GSH), total antioxidant capacity (TAC), and superoxide dismutase (SOD) with an amplification of lipid peroxidation in testes, indicating disruption of the antioxidant defense system. Furthermore, myeloperoxidase (MPO) activity and DNA damage were significantly heightened, implying inflammation and genotoxicity, respectively. Moreover, steroidogenic enzymes, including 17ß-Hydroxy Steroid Dehydrogenase 3 (HSD17b3), 3ß-Hydroxy Steroid Dehydrogenase 2 (HSD3b2), 17α-hydroxylase/17,20-lyase (CYP17A1), and steroid 5α-reductase 2 (SRD5A2) were markedly diminished accompanied with disorders in luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone. Besides, spermatozoa quality was reduced, associated with a diminution in the diameter of seminiferous tubules. By contrast, PRO or ROJ significantly protected and/or counteracted the Cd-induced pathophysiological consequences, ameliorating antioxidant and inflammatory biomarkers, steroidogenic enzymes, hormonal levels, and sperm properties, along with lessening DNA impairments. Critically, histological and ultrastructural analyses manifested several anomalies in the testicular tissues of the Cd-administered group, including the Leydig and Sertoli cells and spermatozoa. Conversely, PRO or ROJ sustained testicular tissues' structure, enhancing spermatozoa integrity and productivity. Interestingly, treatment with PRO or ROJ improved fertility indices through offspring rates compared to the Cd-animal group. Our data suggest that PRO is a more effective countermeasure than ROJ against Cd toxicity for securing the delicate testicular microenvironment for spermatogenesis and steroidogenesis.

Reversal Effects of Royal Jelly and Propolis Against Cadmium-Induced Hepatorenal Toxicity in Rats.

Heavy metal toxicity is an exponentially growing health problem. In this study, we aimed to assess the protective properties of propolis and royal jelly against cadmium adverse effects. Thirty-two adult male rats were included in our study; kidney and liver functions, histopathological changes, and the level of oxidative stress were evaluated in rats exposed to a daily dose of 4.5 mg cadmium per kilogram of body weight for 1 month and those cotreated simultaneously with either propolis (50 mg/kg/day) or royal jelly (200 mg/kg/day) with cadmium compared to control animals. Cadmium-mediated hepatorenal toxicity was manifested as per the increased oxidative stress, function deterioration, and characteristic histopathological aberrations. The supplementation of royal jelly or propolis restores most of the affected parameters to a level similar to the control group. However, the parameters describing the grade of DNA damage and the interleukin-1ß expression in the liver, as well as the levels of malondialdehyde and metallothionein, were slightly elevated compared to controls, despite the regular use of royal jelly or propolis. It is worth noting that better results were found in the case of royal jelly compared to propolis administration. Most likely, the ability of both products to chelate cadmium and contribute in reducing oxidative stress is of great importance. However, further investigations are needed to complement the knowledge about the expected nutritional and medicinal values.

Sericin and melatonin mitigate diethylnitrosamine-instigated testicular impairment in mice: Implications of oxidative stress, spermatogenesis, steroidogenesis, and modulation of Nrf2/WT1/SF-1 signaling pathways.

AIMS: This study aimed to investigate the therapeutic influence of combination therapy with sericin and melatonin on attenuating diethylnitrosamine (DEN)-instigated testicular dysfunction in mice and defining the molecular mechanisms involved in orchestrating redox signaling pathways and restoring spermatogenesis and steroidogenesis. MATERIALS AND METHODS: Different groups of male Swiss albino mice were established and injected with respective drugs intraperitoneally. Semen analysis, hormonal assays, and oxidative stress biomarkers were evaluated. Additionally, melatonin and its receptors, WT1, SF-1, vimentin, Nrf2, and ANXA1 expressions were assessed. Histopathological and ultrastructural features of the testes were investigated by semithin, SEM, and TEM analyses. KEY FINDINGS: Exposure to DEN exhibited pathophysiological consequences, including a remarkable increase in lipid peroxidation associated with substantial diminutions in SOD, CAT, GPx, GSH, GSH:GSSG, and GST. Furthermore, it disrupted spermatozoa integrity, testosterone, FSH, LH, melatonin, and its receptors (MT1 and MT2) levels, implying spermatogenesis dysfunction. By contrast, treatment with sericin and melatonin significantly restored these disturbances. Interestingly, the combination therapy of sericin and melatonin noticeably augmented the Nrf2, WT1, and SF-1 expressions compared to DEN-treated mice, deciphering the amelioration perceived in antioxidant defense and spermatogenesis inside cells. Furthermore, immunohistochemical detection of ANXA1 alongside histopathological and ultrastructural analyses revealed evident maintenance of testicular structures without discernible inflammation or anomalies in mice administered with sericin and melatonin compared to the DEN-treated group. SIGNIFICANCE: Our findings highlighted that treatment with sericin and melatonin alleviated the testicular tissues in mice from oxidative stress and dysregulated spermatogenesis and steroidogenesis engendered by DEN.

Adaptation by death? Cell death-based tolerance to cadmium in 150-generation exposure of Spodoptera exiqua Hübner (Lepidoptera: Noctuidae).

Mechanisms, including autophagy and apoptosis, which serve to regulate and ensure proper organism functions under optimal conditions, play additional defensive roles under environmental pressure. The aim of this study was to test the following hypotheses: (i) elevated autophagy and apoptosis intensity levels, as defensive processes in response to contact with cadmium, are maintained for a limited number of generations and (ii) the number of generations after which levels of cell death processes reach the reference level depends on selective pressure. Cell death processes were assessed by light and transmission electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL), and cytometric analyses. Model insects (Spodoptera exiqua, Hübner, 1808) were orally exposed to various concentrations of cadmium for 18 generations and compared with reference strains exposed to cadmium or not (control) for over 150 generations. Elevated programmed cell death intensity levels decreased after several generations, indicating tolerance of individuals to cadmium in the diet and verifying the first hypothesis; however, testing the second hypothesis indicated that the number of generations depended not only on pressure intensity, but also on cell death type, since levels of autophagy remained increased for a minimum of 12 generations.

Joint cadmium and polypropylene microparticle action in cadmium tolerant model insect.

Microplastic enlisted as a contaminant of emerging concerns in polluted environments interact with "traditional" contaminants such as metals, causing, among others, their increased accumulation in the body. Harmful effects depend on the exposed animals' possible preadaptation and/or cross-tolerance. The project aimed to assess the role of this phenomenon in the limited toxicity of polypropylene fibers (PPf) in 0%, 0.02%, 0.06, 0.18%, 0.54%, and 1.6% of Cd-supplemented food of larvae of Spodoptera exigua multigenerationally selected to cadmium tolerance. The activity of 20 digestive enzymes (API-ZYM test), defensins, and heat shock proteins, HSP70 levels in the exposed groups were used as biomarkers. PPfs caused the increase of Cd accumulation in the body, while intake of polypropylene microfibers did not change the biomarker levels. Moreover, multigenerational Cd pre-exposure, due to increased tolerance of Cd and, possibly, cross-tolerance, prepares the insects for an additional stressor (PPf) alone and in interaction with cadmium.

Reduced body length and morphological disorders in Chrysomya albiceps (Diptera: Calliphoridae) larvae reared on aluminum phosphide-treated rabbits.

Assessing the time of death based on the growth and development of insects is a critical task in forensic entomology. The rate of larvae development can be affected by a variety of toxins, including pesticides. Aluminum phosphide (AlP) is a low-cost insecticide that has yet to be tested for entomotoxicological significance, despite the fact that it is frequently the cause of fatal poisoning. In this study, we measured the body length of Chrysomya albiceps larvae reared on the carcasses of rabbits poisoned with AlP and analyzed the morphological changes of the larvae reared on the carcasses of rabbits poisoned with AlP. The concentration of AlP in the body of the larvae was significantly lower than in rabbit tissues. Insects from the AlP group had a significantly lower gain in body length. Furthermore, deformities in the larvae were found. Smaller respiratory spiracles were found, as well as a deformed small posterior end with hypogenesis of the posterior respiratory spiracles. Thus, disturbed growth and development of carrion flies found at a crime scene could indicate pesticide poisoning, such as aluminum phosphide.

UPLC-MS/MS Analysis of Naturally Derived Apis mellifera Products and Their Promising Effects against Cadmium-Induced Adverse Effects in Female Rats.

Honeybee products arouse interest in society due to their natural origin and range of important biological properties. Propolis (P) and royal jelly (RJ) attract scientists' attention because they exhibit antioxidant, anti-inflammatory, anti-bacterial, anti-tumor, and immunomodulatory abilities. In this study, we tested whether P and RJ could mitigate the adverse effects of cadmium (Cd) exposure, with particular emphasis on the reproductive function in female rats. In this line, one week of pretreatment was established. Six experimental groups were created, including (i) the control group (without any supplementation), (ii) the Cd group (receiving CdCl2 in a dose of 4.5 mg/kg/day), (iii) the P group (50 mg of P/kg/day), (iv) RJ group (200 mg of RJ/kg/day), (v) P + Cd group (rats pretreated with P and then treated with P and Cd simultaneously), (vi) RJ + Cd group (animals pretreated with RJ before receiving CdCl2 simultaneously with RJ). Cd treatment of rats adversely affected a number of measured parameters, including body weight, ovarian structure and ultrastructure, oxidative stress parameters, increased ovarian Cd content and prolonged the estrous cycle. Pretreatment and then cotreatment with P or RJ and Cd alleviated the adverse effects of Cd, transferring the clusters in the PCA analysis chart toward the control group. However, clusters for cotreated groups were still distinctly separated from the control and P, or RJ alone treated groups. Most likely, investigated honeybee products can alter Cd absorption in the gut and/or increase its excretion through the kidneys and/or mitigate oxidative stress by various components. Undoubtedly, pretreatment with P or RJ can effectively prepare the organism to overcome harmful insults. Although the chemical composition of RJ and P is relatively well known, focusing on proportion, duration, and scheme of treatment, as well as the effects of particular components, may provide interesting data in the future. In the era of returning to natural products, both P and RJ seem valuable materials for further consideration as anti-infertility agents.

Stage-, sex- and tissue-related changes in H2O2, glutathione concentration, and glutathione-dependent enzymes activity in Aiolopus thalassinus (Orthoptera: Acrididae) from heavy metal polluted areas.

This study is part of a large project carried out at the Cairo University, Egypt, and focused on assessing physiological and biochemical changes in Aiolopus thalassinus under the influence of environmental pollution with heavy metals (Pb, Cd, Cu, and Zn). The study aimed to investigate parameters related to maintaining redox balance, with particular emphasis on stage-, sex- and tissue-dependent differences in H2O2 and glutathione (GSH) levels and activity of selected enzymes involved in GSH metabolism. A noticeable increase in the concentration of H2O2 was found, especially in the gut of 5th instar nymphs and females from the highly polluted site. An increase in GSH concentration was significant, especially in the gut of adult A. thalassinus from the high polluted site. However, recycling of reduced form of glutathione in the gut by glutathione reductase (GR) was relevant only for females from the high polluted site. Nymphs and females generally showed higher glutathione S-transferase (GST) activity, especially in the gut. These stage- and sex-related differences can result from different growth dynamic and various reproductive functions of nymphs and both sexes. The digestive track is in direct contact with xenobiotics consumed with food. Nymphs are characterized by vigorous growth, they feed intensively, and their development processes are associated with substantial oxygen consumption. Also, maintaining the antioxidant system at a high level can be more important for females than males due to egg production over a long period. It appears that de novo GSH synthesis is a favorable and cost-effective adaptation mechanism for A. thalassinus living in the high polluted site.

Energy reserves, oxidative stress and development traits of Spodoptera exigua Hübner individuals from cadmium strain.

Cadmium as a common environmental stressor may exert highly toxic effects on herbivorous insects. The question was whether possible elevation of an oxidative stress and imbalance of energetic reserves in insects may depend on developmental stage, sex and insect population's multigenerational history of exposure to cadmium. So, the aim of this study was to compare of the development traits, total antioxidant capacity, lipid peroxidation, RSSR to RSH ratio and the concentration of carbohydrates, glycogen, lipids and proteins in whole individuals (larvae or pupae) of Spodoptera exigua originating from two strains: control and selected over 120 generations with sublethal metal concentration (44 Cd mg per dry weight of diet). Generally, the increase of the protein, carbohydrates, glycogen concentration and lipid peroxidation decrease with age of the larvae were found. Revealed cases of a higher mobilisation of carbohydrates and proteins, and changes in total antioxidant capacity or lipid peroxidation, in individuals being under metal exposure, occurred in strain-depended mode. Short-term Cd exposure effect was connected with possible higher engagement of proteins and glycogen in detoxification processes, but also higher concentration of lipid peroxidation. In turn, for long-term Cd exposure effect lower lipids concentration and higher thiols usage seemed to be more specific.

Autophagy: a necessary defense against extreme cadmium intoxication in a multigenerational 2D experiment.

Autophagy is a natural process that aims to eliminate malfunctioning cell parts, organelles or molecules under physiological conditions. It is also induced in response to infection, starvation or oxidative stress to provide energy in case of an energy deficit. The aim of this 2-dimensional study was to test if, and if so, how, this process depends on the concentration of cadmium in food (with Cd concentrations from 0 to 352 µg of Cd per g of food (dry weight)-D1 dimension) and the history of selection pressure (160 vs 20 generations of exposure to Cd-D2 dimension). For the study, the 5th instar larvae of a unique strain of the moth Spodoptera exigua that was selected for cadmium tolerance for 160 generations (44 µg of Cd per g of food (dry weight)), as well as 20-generation (11, 22 and 44 µg of Cd per g of food (dry weight)) and control strains, were used. Autophagy intensity was measured by means of flow cytometry and compared with life history parameters: survivability and duration of the 3rd larval stage. The highest values of autophagy markers were found in the groups exposed to the highest Cd concentration and corresponded (with a significant correlation coefficient) to an increased development duration or decreased survivorship in the respective groups. In conclusion, autophagy is probably initiated only if any other defense mechanisms, e.g., antioxidative mechanisms, are not efficient. Moreover, in individuals from pre-exposed populations, the intensity of autophagy is lower.

DNA damage in Spodoptera exigua after multigenerational cadmium exposure - A trade-off between genome stability and adaptation.

Human activity is a serious cause of extensive changes in the environment and a constant reason for the emergence of new stress factors. Thus, to survive and reproduce, organisms must constantly implement a program of adaptation to continuously changing conditions. The research presented here is focused on tracking slow changes occurring in Spodoptera exigua (Lepidoptera: Noctuidae) caused by multigenerational exposure to sub-lethal cadmium doses. The insects received food containing cadmium at concentrations of 5, 11, 22 and 44 µg per g of dry mass of food. The level of DNA stability was monitored by a comet assay in subsequent generations up to the 36th generation. In the first three generations, the level of DNA damage was high, especially in the groups receiving higher doses of cadmium in the diet. In the fourth generation, a significant reduction in the level of DNA damage was observed, which could indicate that the desired stability of the genome was achieved. Surprisingly, however, in subsequent generations, an alternating increase and decrease was found in DNA stability. The observed cycles of changing DNA stability were longer lasting in insects consuming food with a lower Cd content. Thus, a transient reduction in genome stability can be perceived as an opportunity to increase the number of genotypes that undergo selection. This phenomenon occurs faster if the severity of the stress factor is high but is low enough to allow the population to survive.

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.

Microevolution or wide tolerance? Level of stress proteins in the beet armyworm Spodoptera eqigua hübner (Lepidoptera: Noctuidae) exposed to cadmium for over 150 generations.

The aim of this study was to investigate whether the cadmium tolerance developed in the beet armyworm Spodoptera exigua selected for over 150 generations may be related to synthesis of the stress proteins metallothioneins (Mts) and 70 kDa heat shock proteins (HSP70). To achieve this, six S. exigua strains (control, k), 150-generation Cd exposure strain (cd), and four 18-generation Cd exposure strains differing in Cd concentration (cd44, cd22, cd11, cd5) were reared. Stress protein level was measured in the midgut of the 5th larval stage after 1-6, 12 and 18 generations. Cd contents was measured in the pupae. Unlike Cd concentration, which depended on metal contents in food but was not generation-dependent, the pattern of Mts and HSP70 concentrations changed in experimental strains from generation to generation. Stress protein levels in the insects exposed to the highest Cd concentration (the same as in the 150-generation Cd exposure strain), initially higher than in the control strain, after the 12th generation did not differ from the level measured in the control strains. It seems therefore that stress proteins play a protective role in insects of lower tolerance to cadmium. The tolerance developed during multigenerational exposure probably relies on mechanisms other than Mt and HSP70 synthesis.

Antioxidant enzyme activity in responses to environmentally induced oxidative stress in the 5th instar nymphs of Aiolopus thalassinus (Orthoptera: Acrididae).

The response of antioxidant enzymes to oxidative environmental stress was determined in 5th instar nymphs of Aiolopus thalassinus (Orthoptera: Acrididae) collected from sites with different level of pollution with heavy metals, PO43-, and SO42-. The high polluted site induced higher DNA damage to individuals compared to the control site. The highest values of tail length (TL), tail moment (TM), and percent of DNA in tail (TDNA) were found in the gut of 5th instar nymphs from a high polluted site. Also, protein carbonyls and lipid peroxide levels were significantly higher in insects collected from polluted sites compared to those from the control site. A strong positive correlation between both protein carbonyl and lipid peroxide concentration and the pollution level of the sites was found in all tissues of the insects. The activity of superoxide dismutase (SOD) in the brain of insects collected from the high polluted site was significantly higher than that in the thoracic muscles and gut. We observed strong inhibition of catalase (CAT) activity. This effect was apparently caused by pollutants present at the high polluted site. The level of pollution significantly influenced polyphenol oxidase (PPO) activity in A. thalassinus nymphs in all examined tissues. The highest values were observed in the brain. The relationship between pollution and ascorbate peroxidase (APOX) activity in the examined tissues had no clear tendency. However, the lowest APOX activity was observed in individuals from the low polluted site. Level of pollution of sampling sites, oxidative stress biomarkers, and enzymatic response in A. thalanthsis 5th instar were negatively or positively correlated. Oxidative damage parameters, especially the percent of severed cells, lipid peroxides, and the activity of APOX, can be perceived as good markers of environmental multistress.

Reproduction and development of Spodoptera exigua from cadmium and control strains under differentiated cadmium stress.

The growth and development of living organisms is programmed in genes, but exogenous factors (e.g. cadmium) may modulate endogenous information. Heavy metals may disturb physiological functions and accumulate in the tissues. The insects under prolonged heavy metal stress show some modifications in their metabolism management. The aim of this study was to compare the reproduction and development between individuals of S. exigua from the strain, exposed over 130 generations to sublethal concentration of cadmium (44 mg Cd/kg dry weight of larval diet), and the individuals from the control strain, both additionally exposed to different concentration of cadmium (22-704 mg Cd/kg dry weight of larval diet). The exposure to various cadmium concentrations in the diet revealed survival difference between the cadmium and the control animals at the larvae stage. The differences between adults were not evident. The telomere length (responsible for the duration of a lifespan) in the cadmium strain was shorter in the females than in the males and the individuals from the control strain. TERF1 gene expression (indirectly responsible for the telomere length) was higher in the individuals from the cadmium strain 24 hrs after eclosion. The significant reduction in the larvae body mass was observed in both strains, when the metal concentration was equal to or higher than 264 mg/kg dry weight of larval diet. The EC50 values (defined as of body mass loss), calculated 48 hours after cadmium exposure of individuals from control and cadmium strains, were respectively 632 and 725 mg Cd/kg dry weight of diet. However, some difference in reproduction (the total number of eggs laid and the oviposition time) between the strains appeared only in the groups fed on the uncontaminated diet. The control females laid almost two times more eggs than those from the cadmium strain, and the control ones had more than two times longer oviposition time than the females from the cadmium strain. The fluctuation was also noted in the size of eggs and the hatching success on the following days when both strains were compared, while the hatching success was higher for the insects from the cadmium strain. In conclusion, the insects from the cadmium strain are more resistant to cadmium contamination, as it is evidenced by the EC50 parameter. However, the females from the cadmium strain start laying eggs statistically later, have shorter telomeres and slightly reduced TERF1 gene expression, but hutching success in the strain is significantly higher when compared with the control individuals.

Molecular changes in vitellogenin gene of Spodoptera exigua after long-time exposure to cadmium - Toxic side effect or microevolution?

The reproduction of pest insects is a continuously ongoing issue, especially in the environmental pollution context. Natural or artificial stressing factors enforce a kind of trade-off, most often between growth/survival and reproduction, which improves fitness of the organism. Harmful factors, such as cadmium, can affect the vitellogenesis leading to reduction of yolk synthesis and egg production. The aim of this study was to assess whether 130-generational selection to cadmium in food might have induced change in vitellogenesis of Spodoptera exigua. We analyzed the level of Vg gene expression in S. exigua from the control and the cadmium strain at regular time intervals within 48h after eclosion. The full sequence of Vg gene was also compared between strains. The vitellogenin gene expression in both strains was time-dependent. This dependence was more visible in the control strain. In the cadmium strain the vitellogenin expression was significantly lower, comparing with the control strain in the first day after eclosion but increased significantly in the second day. The sequenced CDS (5286bp long) of the control and the cadmium strains were translated into protein sequences containing both 1761 aa. The protein sequences comparison revealed that there is one amino acid change at aa position 1282. Multiple alignments of six orthologous proteins from different species showed that amino acid change is located in the conserved position. Long-lasting exposure to cadmium resulted in permanent mutation in vitellogenin gene. We do not know yet if the mutation can improve fitness of the cadmium-selected insects. However, we can suppose that the mutation is neutral or even beneficial. The mutation and most probably additional effects of cadmium exposure have an influence on the vitellogenin expression. Some modification in the expression of the vitellogenin receptor are also likely to be important.

Cross tolerance in beet armyworm: long-term selection by cadmium broadens tolerance to other stressors.

Long lasting exposure of animals to stressing factor may lead to the selection of population able to cope with the stressor at lower cost than unexposed individuals. The aim of this study was to assess whether 130-generational selection of a beet armyworm to cadmium in food might have induced tolerance also to other stressors. The potential tolerance was assessed by means of unspecific stress markers: HSP70 concentration, DNA damage level, and energy budget indices in L5 larval instars of beet armyworm. The animals originated from Cd-exposed and control strains exposed additionally in a short-term experiment to high/low temperature or pesticide-spinosad. The application of the additional stressors caused, in general, an increase in the levels of studied parameters, in a strain-dependent manner. The most significant increase was found in HSP70 level in the individuals from the Cd-strain exposed to various spinosad concentration. Therefore, multigenerational contact with cadmium caused several changes that enable the insect to survive under a chronic stress, preparing the organism to the contact with an additional, new stressor. This relationship may be described as a sort of cross tolerance. This may, possibly, increase the probability of population survivorship and, at the same time, decrease the efficiency of pesticide-based plant protection efforts.

Evaluation of Candidate Reference Genes for Quantitative Gene Expression Analysis in Spodoptera exigu a after Long-time Exposure to Cadmium.

Studies on the transcriptional control of gene expression play an important role in many areas of biology. Reference genes, which are often referred to as housekeeping genes, such as GAPDH, G3PDH, EF2, RpL7A, RpL10, TUBα and Actin, have traditionally been assumed to be stably expressed in all conditions, and they are frequently used to normalize mRNA levels between different samples in qPCR analysis. However, it is known that the expression of these genes is influenced by numerous factors, such as experimental conditions. The difference in gene expression underlies a range of biological processes, including development, reproduction and behavior. The aim of this study was to show the problems associated with using reference genes in the qPCR technique, in a study on inbred strains of Spodoptera exigua selected toward cadmium resistance. We present and discuss our results and observations, and give some recommendations concerning the use and limitations of housekeeping genes as internal standards, especially in research on insects. Our results suggest that holometabolism and poikilothermia, as well as time since metamorphosis and the level of exposure to the selective factor (cadmium in this case), have a significant effect on the expression of reference genes.