| dc.description.abstract | INTRODUCTION: Environmental temperature has a direct impact on the development of phytophagous insects, and an indirect, through influence on their host plant composition. Alkaline phosphatases (ALP) and total acid phosphatases (tot ACP) are midgut enzymes included in metabolic processes. Previous contact of population with various stressors, but also their ability to overcome the effects of the raised temperature (thermotolerance), can modify the response of these enzymes to increased environmental temperature.
OBJECTIVES: Our aim was to compare the differences in responses of midgut ALP and tot ACP and expression of their isoforms to increased environmental temperature with and without induced thermotolerance, in gypsy moth 5th instar caterpillars from unpolluted and polluted habitats.
METHOD / DESIGN: Caterpillars were hatched from egg masses collected in unpolluted (UP population) and polluted forest (PP population). They were reared at 23°C (PP23 and UP 23) and 28°C (PP28 and UP28) until 3rd day of 5th instar. In both populations a group of individuals were exposed to 28°C for 24 h (induced thermotolerance) at the beginning of the 4th instar. Afterwards they were returned to 23°C until the sacrification (PP23In and UP23In) or exposed to 28°C for 72h before sacrification on the 3rd day of 5th instar (PP28In and UP28In). The activity of enzymes was measured spectrophotometrically, using p-nitrophenyl phosphate (pNPP) as substrate, under alkaline conditions for ALP and acid conditions for tot ACP. Isoforms of both enzymes were detected on 12% polyacrylamide gel native PAGE.
RESULTS: In UP groups, midgut ALP showed increased activity upon exposure to 28°C, with and without induced thermotolerance, while in PP caterpillars induced thermotolerance was the only factor that elevated ALP activity. Two way ANOVA analysis revealed that the interaction of temperature treatments and population origin (unpolluted vs polluted forest) was extremely significant (F3,67=27.6, p<0.0001) for changes in midgut ALP activity, as well as the individual influence of increased temperature (F3,67=30.9, p<0.0001) and the origin of the population (F1,67=28.6, p<0.0001). Three ALP isoforms were detected. Isoform 1 was present only in PP groups exposed to 28°C, second is present in all experimental groups, and the third showed lower band density in PP treatments in comparison to UP. In UP23In tot ACP activity was elevated, while in PP treatments it was decreased. The interaction of temperature and population origin was extremely significant for tot ACP activity (two way ANOVA, F3,72=10.48, p<0.0001), while their individual influence was not. Four isoforms of tot ACP were detected on gel. Isoform 1 was present only in PP groups, isoform 2 has higher density in both populations and all treatments in comparison to controls. High band density of isoform 3 is present in all experimental groups, while induced thermotolerance and increased temperature, in both population, increased band density of isoform 4.
CONCLUSIONS: Increased environmental temperature and induced thermotolerance have different effects on the activity of both enzymes in caterpillars from unpolluted and polluted habitats. ALP activity was more sensitive to thermal treatments in individuals originating from unpolluted forests, in comparison to those from polluted habitats, where, on the other hand, completely new isoform was detected upon exposure to increased temperature. Tot ACP activity was decreased in all treatments in caterpillars from polluted habitats and a new isoform band was detected on native gels, while in those from unpolluted forest, induced thermotolerance had the effect on the activity of tot ACP. Obtained results indicate the differences in sensitivity to increased environmental temperature between populations with different histories of exposure to pollution and that they must be considered as well. | sr |
