Komórkowe strategie reakcji pająków na stres środowiskowy

Abstract

The aim of the work was to compare cellular effects of natural (heat shock and starvation) and anthropogenic (organophosphorous pesticide) stressors in two, behaviorally different spider species: web building Agelena labyrinthica and wandering Xerolycosa nemoralis, from areas variously polluted by heavy metals. It was crucial to check whether the response of female and male spiders, chronically exposed to high metal concentrations in their habitats to additional stressors, is similar as in spiders from the slightly polluted site and decide which of the stressogenic factors exerts the strongest additional costs for an organism. Since they are the midgut glands that play a strategic role in digestion and detoxification, the analyses were conducted in this organ. Using cytometric techniques and spectrophotometric methods, the following parameters were measured: — the intensity of apoptotic and necrotic changes as well as the quantity of cells with depolarized mitochondria and the caspase-like protease activity level; — the quantitative changes in the cells that positively react with metallothionein (Mt) and heat shock protein (Hsp70) antibodies; — the level of selected antioxidative parameters (total glutathione concentration, activity of selene- -dependent glutathione peroxidase; GPOX, selene- independent glutathione peroxidase; GSTPx, glutathione S-transferase; GST, superoxide dismutase; SOD, catalase; CAT). Obtained results demonstrated that the individuals from heavily polluted areas were more sensitive to applied stressing factors than the animals that have not been exposed to environmental pollutants. The diversity in the cellular response of female and male spiders from variously polluted habitats confirms the necessity of considering the aspect of gender in this kind of comparisons. Mixing males and females together may conceal quantitative correlations between analysed parameters, when coping with long lasting pre-exposure. From among all stressing factors applied, the most pronounced changes, measured as the percentage of apoptotic cells in spider midgut glands, were caused by hunger. Pro-apoptotic effect of the exposure to the applied factors diminished according to the following order: hunger > heat shock + dimethoate > dimethoate > heat shock. Female X. nemoralis seemed more resistant to starvation, since the stressing factor did not enhance necrotic changes in the cells of their midgut glands. In heat shock and/or dimethoate exposed groups the degree of necrotic changes in the organ was higher than in respective A. labyrinthica groups. This may indicate higher sensitivity of the wandering spider species to the remaining stressing factors. Enzymatic neutralization of reactive oxygen species in female X. nemoralis was revealed mainly by the increased activity of SOD and CAT. In spiders also glutathione concentration and activity of glutathione- dependent enzymes were elevated in response to stressing factors. Antioxidative responses registered in A. labyrinthica midgut glands were poorly differentiated and appeared mainly as increased glutathione concentration and gender-related GPOX (females and males) or GSTPx (males) activity. The exposure to stressing factors resulted also in the induction of stress proteins in the spiders. Female X. nemoralis had an increased Mt synthesis while males, under the same conditions, had higher Hsp70 production. In case of female A. labyrinthica stressing factors stimulated the Hsp70 synthesis, while in males both Hsp70 and Mt production was enhanced. In case of wolf spiders (X. nemoralis) the cytoprotective role of SOD and CAT may be verified by positive correlations found for their activity and necrosis and/or apoptosis intensity in the organ. In case of tunnel spiders (A. labyrinthica) both parameters were either negatively correlated or there were no correlations at all. Cytoprotective role in A. labyrinthica is likely played by Hsp70, since the percentage of cells dying according to necrotic pathway was lower in those groups where higher percentage of Hsp70 positive cells was found. Analysed indices of cell death in case of both spider species are good biomarkers of general stress, caused by the exposure to both natural and anthropogenic factors. However, the lack of unequivocal correlations between the level of measured parameters and the kind of stressor is the reason why it is impossible to point out precisely the cause of the changes, but only to confirm the stress itself that, dependently on its intensity, increased apoptotic and/or necrotic changes. The analyses of quantitative ratios may be used in the comparisons of sensitivity of the species to applied stressing factors. Obtained results confirmed inter-species differences in antioxidative strategies found in previous investigations on spiders. Irrespectively of the type of stressor, in case of X. nemoralis an important defensive role in midgut glands is played by SOD and CAT while in case of A. labyrinthica — glutathione and Hsp70. Quantitative changes in the Mt-positive cells in particular experimental groups may indicate that in case of spiders the proteins contribute rather to antioxidative defense than in direct metal binding.