The current study extends our previous findings concerning the sensitivity of Lymantria dispar larvae to cadmium in light of ecotoxicological risk assessment. Here we report the results of the comet assay performed for the first time on this species. We examined the chronic effects of two cadmium concentrations (50 and 100 μg Cd/g dry food) on DNA integrity and haemocyte viability, as well as on fitness-related traits (larval mass and development duration parameters). All parameters were assessed individually and then used to calculate the integrated biomarker response (IBR) index. Egg-masses of L. dispar were collected from two locations in Serbia - the uncontaminated Homolje mountains and a metal-polluted area near Bor copper mines, smelter and refinery. Distinctive patterns in the response of these populations to cadmium exposure were noticed. In haemocytes of larvae from the pollution-free location both cadmium treatments increased the level of DNA damage, although in a similar range. Haemocyte viability and larval mass were reduced, while duration of the fourth instar and total development time were prolonged in a concentration-dependent manner. Cadmium tolerance was noticeable in the population from the metal-contaminated site at all organizational levels. Nevertheless, haemocyte viability in that population was reduced by the stronger treatment. Haemocyte viability was recognized as a promising biomarker due to the evident response of both populations to dietary cadmium. Genotoxicity, fitness-related traits and the IBR index could be used for biomonitoring of sensitive populations not previously exposed to metals.

This paper examined the gut digestive enzymes, trypsin and leucine aminopeptidase (LAP), in gypsy moth (Lymantria dispar, L.) larvae exposed to cadmium. We analyzed the 3-day acute effect, chronic effects from hatching until sacrifice, and recovery from long-term dietary treatment with cadmium concentrations of 10 and 30 μg Cd/g dry food. The activities of both examined enzymes declined at the higher level of cadmium after both acute and chronic treatments and did not recover within 3 days of feeding a diet with no added cadmium. Leucine aminopeptidase was more sensitive because its activity was inhibited after both short-term treatments. Three trypsin and one leucine aminopeptidase isoform were detected by electrophoresis. Egg hatches (full-sib families) differed in enzyme activities, index of phenotypic plasticity, and isozyme expression after different treatments. Statistically significant positive correlations between these enzymes pointed to common genetic regulation. Moreover, variances were higher for the control group than for cadmium treatment groups implying that these proteolytic enzymes did not participate directly in detoxification. These results suggest that, with additional research to discover the mechanisms of enzyme inhibition, trypsin and leucine aminopeptidase might be used as biomarkers to indicate the severity of gastrointestinal disease due to cadmium intake.

The development of “green” alternatives to chemical pesticides could play a crucial role in integrated pest management (IPM). Their use is considered either as a substitution for or in addition to hazardous synthetic products. We analysed the influence of three concentrations of tansy (Tanacetum vulgare L.) essential oil (EO), previously characterised by GC-MS, on the survival and moulting of the 2nd instar and the nutritional indices of the 4th instar gypsy moth (Lymantria dispar L.) larvae. In a residual contact toxicity assessment, the exposure to tansy EO caused low mortality (< 10%) while larval development was significantly slowed down, i.e., the percentage of larvae that moulted into the 3rd instar was reduced. On the other hand, when tansy EO was incorporated into the diet (digestive toxicity assay), high mortality and a lack of moulting after 120 h of eating were recorded for the highest applied concentration of EO. During 48 h of feeding on EO-supplemented food at concentrations of 0.5 and 1% (v/v), the relative growth rate (RGR) of the 4th instar larvae significantly decreased, which can be explained by a significant reduction of the relative consumption rate (RCR) and significantly or marginally significantly lower efficiency of conversion of ingested food into insect biomass (ECI). Although the RCR was also reduced with the lowest applied EO concentration (0.1%), the ECI was not affected which meant the RGR was as high as it was for the control larvae. ECI changes, when two higher EO concentrations were applied, were due to a reduction in the efficiency of conversion of digested food into biomass (ECD), while approximate digestibility was unaffected by the presence of EO in the food. Our results on the significant negative effects of tansy EO on gypsy moth larval survival, development time, and nutritional physiology suggest that it could be considered in future designs for botanical insecticides for gypsy moth control.

Cadmium, like many other pollutants, is nondegradable and can be accumulated by Lymantria dispar at a level that affects fitness components, physiology, and development, which could indicate presence of environment pollution by heavy metals. The cadmium effect on fitness-related traits in the third, fourth, fifth, and sixth instar of L. dispar L. was determined. Furthermore, activities of the following antioxidative defense components after the larvae had been fed on the artificial cadmium-supplemented diet (50 μg Cd/g dry food) were assessed: superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), total glutathione amount (GSH), glutathione-S-transferase (GST), glutathione reductase (GR), and the amount of free sulfhydryl (SH) groups. Statistically significant delay of development in the fourth, fifth, and sixth instar and decrease of the larval mass in the third and fourth instar were estimated after the exposure to cadmium through food in comparison to the control. There were no changes in SOD activity of cadmium-treated larvae. Significantly lower CAT, APOX, and GR activities were recorded in the third, fifth, and in the third instar, respectively. At the same time, higher activity was recorded in the sixth instar, while GST activity was higher in the third. GSH content was significantly lower during all instars after treatment but the amount of SH groups was higher in older larvae. The strategy of antioxidative defense and the adjustment or modulation of fitness-related traits in presence of cadmium was dependent on the age of larvae in L. dispar, which might be used in early metal risk assessment in Lepidoptera and other insects.

The effects of different concentrations of benzo(a)pyrene (BaP) (2, 10, 20, 100, 200, and 2000 ng/g dry food weight) on the activity of acetylcholinesterase (AChE) and concentration of heat-shock protein 70 (hsp70) in fifth instar Lymantria dispar brain tissue were investigated. Inhibition of AChE is a general effect biomarker at the individual level. We observed the most efficient inhibition of this enzyme at medium and high BaP concentrations (20, 100, 200, and 2000 ng/g dry food weight). Western blot analysis revealed the presence of two hsp 70 isoforms, with changed amounts depending on the BaP concentration. A fluctuating response in hsp70 concentration to ingested BaP was observed: with decreased levels in the groups fed 2 and 100 ng BaP/g dry food weight, but increased concentrations in the groups given 10 and 2000 ng/g dry food weight. Even though we detected changes in AChE activity and hsp70 concentration in L. dispar brain tissue upon exposure to different BaP concentrations, we cannot characterize them as sensitive and reliable biomarkers for this xenobiotic in L. dispar caterpillars.

This study aimed to examine the effects of ubiquitous polycyclic aromatic hydrocarbon fluoranthene, supplemented to an artificial diet, on the fitness-related traits and activity of midgut antioxidative enzymes—superoxide dismutase (SOD) and catalase (CAT), and expression of their isoforms in the fifth-instar gypsy moth Lymantria dispar L. Prolonged duration of development and reduced weight and relative growth rate were recorded in larvae reared on the diets supplemented with different concentrations of fluoranthene. SOD and CAT activities were significantly higher in the midguts of fluoranthene-treated larvae, compared to that of the control group. Different expression patterns were detected for SOD as well as for CAT isoforms, depending on the supplemented concentration of fluoranthene. Obtained results suggest that the activity of these enzymes in gypsy moth larvae may be used as biomarkers for assessing pollution, even at low concentrations of the pollutant.

We studied the response of gypsy moth (Lymantria dispar (Linnaeus) (Lepidoptera: Lymantriidae)) to the content of manganese in food in the laboratory breeding of caterpillars. The food of the caterpillars {Betula pendula Roth (Fagales: Betulaceae) leaves} was contaminated by dipping in the solution of MnCl₂·4H₂O with manganese concentrations of 0, 0.5, 5 and 10 mg ml⁻¹, by which differentiated manganese contents (307; 632; 4,087 and 8,124 mg kg⁻¹) were reached. Parameters recorded during the rearing were as follows: effect of manganese on food consumption, mortality and length of the development of caterpillars, pupation and hatching of imagoes. At the same time, manganese concentrations were determined in the offered and unconsumed food, excrements, and exuviae of the caterpillars, pupal cases and imagoes by using the AAS method. As compared with the control, high manganese contents in the food of gypsy moth caterpillars affected the process of development particularly by increased mortality of the first instar caterpillars (8 % mortality for caterpillars with no Mn contamination (T0) and 62 % mortality for subjects with the highest contamination by manganese (T3)), by prolonged development of the first–third instar (18.7 days (T0) and 27.8 days (T3)) and by increased food consumption of the first–third instar {0.185 g of leaf dry matter (T0) and 0.483 g of leaf dry matter (T3)}. The main defence strategy of the caterpillars to prevent contamination by the increased manganese content in food is the translocation of manganese into frass and exuviae castoff in the process of ecdysis. In the process of development, the content of manganese was reduced by excretion in imagoes to 0.5 % of the intake level even at its maximum inputs in food.