Starvation and cadmium affect energy reserves and oxidative stress in individuals of Spodoptera exigua.

Different factors, such as starvation and metal exposure, may affect development and cause oxidative stress in insects. Some host plants may contain a high concentration of cadmium due to their hyperaccumulating property. The negative effects of metals and hunger may be manifested by low availability of energetic substrates. This study aimed to assess whether the insect population with a history of long metal exposure may better manage metal stress or/and starvation at different developmental stages, with the use of energetic substrates. Two strains of Spodoptera exigua model organism were tested: control strain and cadmium strain (treated continuously for over 200 generations with subtoxic amounts of cadmium). The effects of different factors, individually and in combination, on the tested strains were assessed, first by determining the body weight of larvae and pupae and then by estimating the concentration of biomolecules (proteins, carbohydrates, lipids, or glycogen) in the 4th and 5th larval stages and in pupae, and the total antioxidant capacity and lipid peroxidation level in the 4th larval stage. Compared to control strain, cadmium strain individuals exhibited changes in the concentration of soluble carbohydrates and protein. This was partly related to earlier 1-day starvation. In particular, changes in carbohydrate concentration seemed to be a sensitive biomarker of metal stress, independent of the age of individuals and period of starvation. However, the increase in the total antioxidant capacity and the concentration of lipid peroxidation products in the 4th larval stage under the effect of cadmium was dependent on strain origin.

Level of oxidative stress for the land snail Cepaea nemoralis from aged and bioremediated soil contaminated with petroleum products.

Here, we investigated whether the widely distributed snail Cepaea nemoralis could be used as a suitable sentinel animal for assessing the effects of soil contaminants-petroleum oil derivatives-after years of soil ageing and treatment with a bacterial formulation. Oxidative stress was assessed in the foot and hepatopancreas of C. nemoralis L. exposed to soil contaminated with unleaded petrol, spent engine oil or diesel oil and bioremediated with a bacterial formulation (soil was used 2 years after contamination and bioremediation process). We measured total antioxidant capacity, catalase and glutathione transferase activity and concentrations of superoxide anions, hydrogen peroxide and protein carbonyls in the foot and hepatopancreas of snails after 2 and 4 weeks of treatment. The studied antioxidant responses appeared largely to be tissue and remediation process specific, while the concentrations of superoxide anions, hydrogen peroxide and protein carbonyls depended on time of exposure, tissue type and the type of contaminants, but mostly not on the remediation process. Generally, changes in the concentrations of superoxide anions, hydrogen peroxide and protein carbonyls in the hepatopancreas of snails seemed to be a suitable measure to assess the risk of animals exposed to soil contaminated with petroleum substances and used after many years of ageing and treatment with a microbial formulation.

The effect of selected immunostimulants on hemocytes of the false black widow Steatoda grossa (Theridiidae) spiders under chronic exposition to cadmium.

The aim of this study was to analyze whether, and to what extent, long-term exposure to cadmium, administered in sublethal concentrations by the oral route, caused changes in the immune potential of hemocytes in adult female Steatoda grossa spiders. We used artificial and natural immunostimulants, namely phorbol 12-myristate 13-acetate (PMA) and bacterial cell suspension based on Gram-positive (G+, Staphylococcus aureus) and Gram-negative (G-, Pseudomonas fluorescens) bacteria, to compare the status of hemocytes in nonstimulated individuals and those subjected to immunostimulation. After cadmium exposure, the percentage of small nongranular hemocytes in response to G+ cell suspension and PMA mitogen was decreased. Furthermore, in the cadmium-intoxicated spiders the percentage of plasmatocytes after immunostimulation remained lower compared to the complementary control group. Exposure to cadmium also induced several degenerative changes, including typical apoptotic and necrotic changes, in the analyzed types of cells. Immunostimulation by PMA mitogen and G+ bacterial suspension resulted in an increase in the number of cisterns in the rough endoplasmic reticulum of granulocytes, in both the control group and cadmium-treated individuals. These changes were accompanied with a low level of metallothioneins in hemolymph. Chronic cadmium exposure may significantly weaken the immune defense system of spiders during infections.

The effects of high-monosaccharide diets on development and biochemical composition of white-eyed mutant strain of house cricket (Acheta domesticus).

Tryptophan (TRP) is one of the essential amino acids in the animal body. Its exogenicity and low concentrations mean that it can be regarded as one of the key regulatory molecules at the cellular as well as physiological level. It has been shown to have a number of essential functions, such as in the production of other biologically active molecules. The main objective of this project was to investigate the effects of a high monosaccharide diet (HMD) on a hemimetabolic insect-house cricket (Acheta domesticus) and a mutant strain with impaired visual pigment synthesis (closely related to the tryptophan and kynurenine (KYN) metabolic pathway)-white eye. This study was aimed at determining the effects of glucose and fructose on cricket development and biochemical composition. A parallel goal was to compare the response of both cricket strains to HMD. ELISA assays indicated dysfunction of the TRP-KYN pathway in white strain insects and an elevated KYN/TRP ratio. Biochemical analyses demonstrated the effects of HMD mainly on fat and glycogen content. A decrease in food intake was also observed in the groups on HMD. However, no changes in imago body weight and water content were observed. The results of the study indicate a stronger response of the white strain to HMD compared to the wild-type strain. At the same time, a stronger detrimental effect of fructose than of glucose was apparent. Sex was found to be a modulating factor in the response to HMD.

Effect of long-term cadmium and copper intoxication on the efficiency of ampullate silk glands in false black widow Steatoda grossa (Theridiidae) spiders.

The aim of the study was to compare cellular effects of xenobiotic cadmium and biogenic copper in ampullate silk glands of false black widow Steatoda grossa spider after long-term exposure via ingestion under laboratory conditions. Both the level of selected detoxification parameters (glutathione S-transferase, catalase, and the level of total antioxidant capacity) and degree of genotoxic changes (comet assay) were determined in the silk glands. Additionally the contents of selected amino acids (L-Ala, L-Pro, L-His, L-Phe, DL-Ile, and DL-Asn) in the hunting webs produced by spiders of this species were assessed. The ability of S. grossa females to accumulate cadmium was higher than that for copper. Long-term exposure of spiders to copper did not change the level of detoxification parameters, and the level of DNA damage in the cells of ampullate silk glands was also low. Cadmium had a stronger prooxidative and genotoxic effect than copper in the cells of the analyzed silk glands. However, regardless of the type of metal used, no significant changes in the level of amino acids in silk were found. The obtained results confirmed the effectiveness of metal neutralization mechanisms in the body of the studied spider species, which results in the protection of the function of ampullate silk glands.

The effect of ingested cadmium on the calorific value and structural properties of hunting webs produced by Steatoda grossa (Theridiidae) spiders.

The study aimed to assess whether cadmium administered via ingestion to Steatoda grossa cobweb spiders (Theridiidae) affects the energy content and selected structural properties of the produced hunting webs. Cadmium content in webs was assessed with AAS and SEM X-ray microanalysis, while the diameters of silk fibers were estimated with SEM. The energy content of samples was measured in an oxygen micro-bomb calorimeter. Females and males showed different reactions to cadmium supplied through food. In comparison to females, males displayed higher metal concentrations in their bodies and hunting webs, however their calorific values and structural features were not significantly changed. Cadmium-treated females spun webs with smaller single-strand diameters and more frequent multi-stranded threads and invested 47% less energy in web production than the control individuals. It cannot be excluded that such a reduction in energy expenditure for web building in females resulted from energetically costly detoxifying reactions triggered in response to direct and indirect effects of cadmium toxicity.

Antioxidative and immunological responses in the haemolymph of wolf spider Xerolycosa nemoralis (Lycosidae) exposed to starvation and dimethoate.

The aim of this study was to assess the intensity of enzymatic antioxidative parameters [catalase (CAT), glutathione peroxidase (GSTPx), glutathione reductase (GR), total antioxidant capacity (TAC)] and percentage of high granularity cells as well as low to medium granularity cells in haemolymph of wolf spiders Xerolycosa nemoralis exposed to starvation and dimethoate under laboratory conditions. Only in starved males, haemolymph included a lower percentage of high granularity cells, accompanied by high activity of CAT and GSTPx, than in the control. Exposure of males to dimethoate increased CAT activity, after single application, and significantly enhanced GR activity, after five-time application. In females, five-time contact with dimethoate elevated the percentage of high granularity cells. As in comparison to females, male X. nemoralis were more sensitive to the applied stressing factors, it may be concluded that in natural conditions both food deficiency and chemical stress may diminish the immune response of their organisms.

X-ray microtomography for imaging of developing spiders inside egg cocoons.

Embryogenesis is especially sensitive to external factors. The changes in its course are often used as biomarkers of environmental impact. Since spider embryogenesis takes place inside cocoons, it is crucial to find a reliable tool to analyze this developmental phase with no intrusion into the cocoons. The aim of this study was to verify the efficacy of X-ray microtomography for non-invasive analysis of embryonic morphology and egg quantity in the cocoons of Xerolycosa nemoralis and Agelena labyrinthica from polluted and reference sites. X-ray microtomography slice images as well as 3D images and animations obtained from digital visualization of those slides were used to study the morphology of embryos and egg arrangement in the cocoons. Any disorders in embryogenesis or malformation of embryos in relation to site of origin have not been found, but inside an egg cocoon of X. nemoralis from the polluted site embryos differing form each other by one developmental stage were identified. Egg calculation revealed a K- reproductive strategy of X. nemoralis from polluted sites. Finally, future prospects and benefits, and weaknessess of this method for the study of spider cocoons have presented.

The effects of cadmium or zinc multigenerational exposure on metal tolerance of Spodoptera exigua (Lepidoptera: Noctuidae).

The effects of ten generational zinc or cadmium pre-exposure on metal tolerance among beet armyworm Spodoptera exigua individuals were compared. These effects were assessed in animals from the 11th generation, reared on a diet either uncontaminated or contaminated with metal (cadmium or zinc). The survival rate of larvae and the degree of metal accumulation (in larvae, pupae and moths; among larval organs: gut and fat body) were analysed. Catalase, superoxide dismutase and glutathione transferase activity in larval organs of individuals subjected to different metal treatments were also measured. Animals transferred from control rearing to metals (cadmium or zinc) in the 11th generation, as well as those from multigenerational zinc treatment, but not from multigenerational cadmium treatment, had a significantly lower survival rate than control animals. Insects from the groups with the high metal treatment had high bioaccumulation factors (above 3.7 and 2.3 following cadmium and zinc, respectively). Cadmium (but not zinc) pre-exposure had a significant effect on metal accumulation in larvae. Multigenerational metal pre-exposure seemed to have mainly a negative effect on glutathione transferase activity in the gut of larvae from the 11th generation, in the case of the individuals exposed to metal other than that used in pre-exposure treatment or kept in control conditions. However, in the case of zinc pre-exposure, such effect was only apparent when zinc was replaced by cadmium. The long-term effect of cadmium on catalase activity in larvae was found.

The reproductive potential of the spiders Agelena labyrinthica and Xerolycosa nemoralis from areas contaminated with metals.

Spiders successfully colonize industrially contaminated environments and maintain relatively stable populations. The aim of this study was to explain the reproductive strategies of two spider species, Xerolycosa nemoralis (an actively hunting, sit-and-pursue predator) and Agelena labyrinthica (a web-building, sit-and-wait predator), between contaminated and uncontaminated sites. Spiders were collected from a reference site (Pilica) and two contaminated sites (Olkusz and Welnowiec). The amount of energy allocated to the eggs and the number of eggs and hatchlings as well as the hatching success were compared. Wolf spiders from the contaminated sites produced fewer but relatively energy-rich eggs, whereas web-building spiders invested their energy in the production of a higher number of less energy-rich eggs. The comparisons of the hatching percentages suggested that in the contaminated habitats, X. nemoralis achieve a hatching success similar to or higher than that of the reference population at Pilica. A. labyrinthica in the contaminated sites invested a larger amount of energy in eggs than at the reference site, but the hatching success found for this species in the contaminated areas was lower than that found at the reference site.

Effects of multigenerational cadmium exposure of insects (Spodoptera exigua larvae) on anti-oxidant response in haemolymph and developmental parameters.

Biochemical and organismal indices of metal tolerance were studied in Spodoptera exigua exposed to a cadmium-contaminated diet for one or many (33 or 61) generations. Reduced and oxidised glutathione, protein thiols, total anti-oxidant capacity level, glutathione transferase activity, and Cd accumulation were assayed in the haemolymph of the last instar larvae. The cadmium concentration in the whole larval body as well as larval survival, larval duration time and last instar body weight were also measured. Elevated cadmium concentration in the whole body, higher mortality and longer duration of the larval stage in one-generation exposed insects in comparison with those exposed for many generations suggest that metal tolerance builds over time. For the larvae from multigeneration metal treatment, the higher cadmium concentration in larval haemolymph positively correlated with glutathione oxidation and total anti-oxidant capacity. One-generation exposed insects had lower metal concentration in haemolymph than did 33-generation exposed insects.

Quantitative immunodetection of metallothioneins in relation to metals concentration in spiders from variously polluted areas.

Spiders inhabiting post industrial environments, such as waste heaps or ore-bearing areas, are exposed to high concentrations of metals, accumulated in the body of their prey and transferred along food chains. Therefore spiders are pressed to develop metal-neutralization strategies. Low-molecular, multifunction proteins: metallothioneins (MTs), often postulated as biomarkers of metal exposure, are known to bind metals and thus protect organisms against their toxic effects. Yet the proteins are still not well recognized in spiders. The aim of this study was to assess, by immunodetection method, ELISA, the concentration of metallothioneins in adult females of three web building spider species: Araneus diadematus (Araneidae), Agelena labyrinthica (Agelenidae) and Linyphia triangularis (Linyphiidae) from three variously polluted areas in southern Poland: Olkusz, ore-bearing post industrial site; Katowice-WeLnowiec: post metallurgic waste heap, Pilica: the reference, rural, area. The concentration of metallothioneins has been analyzed in relation to the metal concentration in spiders body. The study gives the evidence that metallothioneins are reliably detectable by means of ELISA technique. The analysis of results obtained shows a strong species-dependence of the MTs level. Positive correlations between MTs concentration and metal body burden (mainly Zn and Pb) were found. This suggests that the proteins play an important role in the neutralization and regulation of metal ions in spiders. The same correlation indicate the possibility to consider MTs in spiders as biomarkers of metal exposure and effects. However, the species specificity as well as metal characteristics should be taken under account.

Metallothioneins and energy budget indices in cadmium and copper exposed spiders Agelena labyrinthica in relation to their developmental stage, gender and origin.

The aim of our studies was to explain the role of metallothioneins (MTs) in the neutralization of excessive amounts of metals (essential: copper (Cu) and toxic: cadmium (Cd)) and to describe the energy status in metal-exposed spiders Agelena labyrinthica in relation to its developmental stage, gender and origin. Juvenile, female and male spiders were collected from three variously polluted habitats, transferred to the laboratory and exposed to the metals in their diet. Cu and Cd accumulation in the body and exuviae, bioaccumulation factor, percentage of metallothionein positive cells, MT concentration, percentage of cells with depolarized mitochondria, ATP concentration and ADP/ATP ratio were measured and calculated. Cu appeared to be regulated and its excess is eliminated via, among others, the molting process, while Cd was rather accumulated by the spiders. The level of MTs increased significantly mainly in females exposed to both metals, irrespectively of the pollution degree of their site of origin, indicating a defensive role of the proteins. In general, even if both the MT level and the energy status indices were positively correlated with Cd and Cu concentrations in the spider body, the energy status of A. labyrinthica did not seem disturbed.

Selenium modifies glutathione peroxidase activity and glutathione concentration in mice exposed to ozone-provoked oxidative stress.

The aim of this study was to show the direct effect of selenium on glutathione peroxidase (GSH-Px) activity and GSH/GSSG concentrations in 3- and 6-month-old mice. An ozone-oxygen mixture was used to provoke an oxygen stress. To measure the Se-effect mice were gavaged with sodium selenite. GSH-Px activity and total glutathione concentrations were determined in serum and in the postnuclear fraction of liver and lungs. Additionally glutathione concentrations were determined in whole blood. Both ozone and selenium, administered separately, reduced GSH-Px activity in lungs of 6-month-old animals, while in young mice an opposite effect of Se was observed. Ozone administered jointly with Se did not influence GSH-Px activity in 6-month-old mice, while in young, 3-month-old mice, a stimulatory effect in lungs was observed. There were no significant changes in GSH-Px activity in the liver of 6-month-old mice, but the stimulatory effect occurred in young mice treated with Se and Se & ozone jointly. In young mice, ozone (also ozone with Se) augmented glutathione concentrations. The response to ozone and selenium strictly depended on age and the antagonism between selenium and ozone was observed only in a few cases.

Activity of glutathione S-transferase in Spodoptera exigua larvae exposed to cadmium and zinc in two subsequent generations.

The aim of our study was to establish changes in activity of important in detoxification enzyme-glutathione S-transferase (GST): in alimentary tract, fat body and Malpighian tubules of Spodoptera exigua larvae being under cadmium and zinc exposure through the first as well as the second generation. There was registered enhancement of the enzyme activity in the fat body and the Malpighian tubules caused by zinc as well as its decrease in the Malpighian tubules under cadmium action. Amounts of metals in the alimentary tract were either several times higher than in the diet ingested by larvae or than in the fat body. Metal concentration in the fat body correlated with the level of the enzyme activity (positive correlation for zinc and negative for cadmium). The effect of metal action differentiated dependently on time exposition.