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Cadmium (Cd) presence in terrestrial ecosystems is a serious threat that requires continuous development of biomonitoring tools. Ideally, a suitable biomarker of exposure should respond to the toxicant consistently in different populations regardless of previous exposure to pollution. Here we considered the activities and isoform patterns of certain proteases and acid phosphatases (ACP) in the midgut of Lymantria dispar larvae as well as the integrated biomarker response (IBR) for application in Cd biomonitoring. We compared the responses of caterpillars originating from unpolluted and polluted localities after they had been chronically subjected to dietary Cd (50 and 100 μg Cd/g dry food). The population inhabiting the unpolluted forest was far more sensitive to Cd exposure as the activities of total proteases, trypsin (TRY) and leucine aminopeptidase (LAP) were mostly reduced while the activities of total and non-lysosomal ACP were increased. Non-lysosomal ACP activity was elevated in larvae from the contaminated site in response to the higher Cd concentration. Exposure to the metal resulted in numerous alterations in the pattern of enzyme isoforms, but the responses of the two populations were similar except that larvae from the polluted locality were more tolerant to the lower Cd concentration. Non-lysosomal ACP activity and the appearance of ACP isoforms 4 and 5 together with the IBR index are the most promising indicators of Cd presence, potentially applicable even in populations with a history of exposure to pollution. TRY and total ACP activities could be used to monitor populations at uncontaminated localities., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cadmium (Cd) can display a variety of different effects on living organisms. The objectives of the present study were to investigate Cd bioaccumulation and differences in parameters of oxidative stress (activities of the enzymes: superoxide dismutase, catalase, glutathione reductase, ascorbate peroxidase, glutathione S-transferase, and amounts of non-enzymatic free sulfhydryl groups and total glutathione) and cholinesterase activity in larval brain and midgut tissues of the polyphagous forest insect Lymantria dispar collected from unpolluted and polluted oak forests. Fourth instar L. dispar caterpillars from the unpolluted forest had higher body mass but accumulated more Cd in comparison to caterpillars from the polluted forest. In both populations the midgut was more sensitive than the brain to the prooxidative effects of Cd. Enzyme activities and amounts of non-enzymatic parameters tended to be greater in midgut tissues than in the brain, except for cholinesterase activity. Parameters of oxidative stress had higher values in caterpillar tissues from the polluted than from the unpolluted oak forest. The observed differences between the two natural populations point to the importance of knowing the history of population exposure to environmental pollution when monitoring forest ecosystems., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Insects brain as a part of nervous system is the first-line of fast stress response that integrate stress signals to regulate all aspects of insect physiology and behaviour. The cadmium (Cd) bioaccumulation factor (BF), activity of the neurotoxicity biomarker acetylcholinesterase (AChE), dopamine content, expression and amount of Hsp70 in the brain and locomotor activity were evaluated in the 4th instar of Lymantria dispar L. caterpillars fed a Cd supplemented diet and reared in an optimal temperature regime (23 °C) and/or exposed to high temperature (28 °C). The insects originated from two forests, one close to "Nikola Tesla" thermoelectric power plant, Obrenovac (polluted population), and the other Kosmaj mountain (less-polluted population, far from any industrial region). The Cd BF was higher in the less-polluted than in the polluted population especially at the high ambient temperature. AChE activity and dopamine content were changed in the brains of L. dispar from both populations in the same manner. Hsp70 concentration in caterpillar brains showed opposite trends, a decrease in the less-polluted and an increase in the polluted population. Locomotor activity was modified in both Lymantria dispar populations, but the pattern of changes depended on the stressors and their combined effect. ACh activity and dopamine content are sensitive parameters to Cd exposure, regardless of pollutant experience, and might be promising biomarkers in monitoring forest ecosystems., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Activity of glutathione S-transferase (GST) in midgut of gypsy moth caterpillars exposed to 10 and 30μg Cd/g dry food was examined. Based on the enzyme reaction through conjugation with glutathione, overall activity remained unaltered after acute and chronic treatment. No-observed-effect-concentration (10μg Cd/g dry food) significantly increased activity only after 3-day recovery following cadmium administration. Almost all comparisons of the indices of phenotypic plasticity revealed statistically significant differences. Despite the facts that GST has important role in xenobiotic biotransformation, our results indicate that this enzyme in insect midgut does not represent the key factor in cadmium detoxification., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We investigated the effects of acute and chronic treatments with cadmium at 10 μg Cd/g dry food and 30 μg Cd/g dry food on α-glucosidase activity of the 4th instar larvae of Lymantria dispar (Lepidoptera: Lymantriidae) as well as subsequent recovery. Enzyme inhibition was recorded during acute exposure to 30 μg Cd/g dry food and during chronic treatment at the lower metal concentration. After three days recovery from 10 μg Cd/g dry food, the α-glucosidase activity returned to the control level. One-way ANOVA showed that cadmium significantly influenced the activity of α-glucosidase during all treatments. The index of phenotypic plasticity was higher during chronic treatment at 10 μg Cd/g dry food than at 30 μg Cd/g as well as during the recovery. We detected four glucosidase isoforms by NATIVE PAGE. The activities and expressions of the isoforms depended on both larval genotype and cadmium treatment.

Lymantria dispar, as most invasive insect species, is very adaptable and reacts quickly to changing environment. Neuroendocrine system first reacts to stress in insects, and specific neurohormonal reorganization may be used in early heavy metal risk assessment. Prothoracicotropic neurohormones (PTTH) control ecdysteroid synthesis (morphogenetic and stress hormones) in insects. In this article, we report the presence of PTTH immunoreactive molecules in L2' dorsolateral neurosecretory neurons (nsn) in caterpillar brains and changes after exposure to pollutant stress of different intensity. For 3 days, after molting into the 4th instar, caterpillars of Lymantria dispar were fed with a high wheat germ diet without (control) or with added cadmium (experimental groups: 10, 30, 100, 250 μg Cd/g dry food weight). Changes in PTTH producing L2' nsn and differences in the intensity of protein bands in the region of PTTH molecular mass (Mr 11-15 kDa) were analyzed. The number of L2' neurons tended to decrease except in the group given the highest cadmium concentration (250 μg). The neurons were enlarged after acute treatment especially in the group given the highest cadmium concentration. The size of L2' nsn nuclei was decreased only in the group fed with 30 μg Cd. Protein band intensity in the Mr region of PTTH remained unchanged in all groups except for the group given the diet with the highest Cd concentration., (Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.)

Cadmium uptake takes place mainly through food. Lymantria dispar larvae were exposed to dietary cadmium in concentrations of 10 and 30μg Cd/g dry food (NOEC, no-observed-effect and LOEC, lowest-observed-effect concentration, respectively) for acute and chronic treatment and recovery. We established that metal contamination decreased mass only during the chronic treatment at 30μg Cd/dry food with no recovery on removal of cadmium for 3days. Significant reduction of protease activity was detected at LOEC after the acute and chronic treatments. Protease showed enhanced plasticity with regard to the fitness trait (mass) during environmental stress and the higher cadmium load, when it changed. The statistically significant higher index of phenotypic plasticity for protease correlated with lower variability. Protease isoforms at the same cadmium treatments differed between genotypes, while some protease isoforms from one egg-mass differed between cadmium treatments. Owing to the low sensitivity and plasticity of mass change during exposure to cadmium, as well as its small influence, we concluded that larval mass is not a good indicator of cadmium presence in food. We suggest that proteases, with further research, might be a suitable indicator of dietary cadmium contamination, as well as nutriment utilization during heavy metal stress., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Heavy metals damage the structure, chemistry, and function of cells, including enzyme systems inside them. Variation in the profile of biochemical biomarkers in prevalent species should be used for assessing environmental contamination. The present study pays attention to the phosphatases present in the midgut of gypsy moth fourth instar caterpillars, which had been exposed to short- and long-term cadmium intake at 10 and 30 μg Cd/g dry food. Chronic cadmium ingestion significantly inhibited the activity of all examined phosphatases, while only the activity of lysosomal phosphatase was acutely decreased. Total acid phosphatase activity recovered from both long-term cadmium treatments within 3 days. The low index of phenotypic plasticity was connected to high variability of plasticity. Dependence of phosphatase isoforms on genotype and duration of cadmium treatment was determined. We concluded that, with further investigations, profiling of total acid phosphatase activity, as well as the lysosomal fraction can be used as a biomarker for acute sublethal metal toxicity.

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.

Changes in fitness components including larval stage duration, relative growth rate (RGR), and mass of the gypsy moth, Lymantria dispar L. (Lepidoptera: Lymantriidae), were investigated in caterpillars fed a synthetic diet with or without a cadmium supplement (10, 30, 100, 250 microg Cd/g dry food weight). Morphometric changes of large protocerebral dorsomedial A2 neurosecretor neurons, their nuclei and the electrophoresis profiles of brain proteins were analyzed in the 4 instar gypsy moths fed the examined diets. The duration of the fourth larval instars were prolonged and RGR and body mass reduced if the caterpillars were fed diets containing high concentrations of cadmium (100 and 250 microg). The size of large A2 dorsomedial neurosecretory neurons and their nuclei were significantly higher in larvae fed the diets supplemented with 10, 100 and 250 microg Cd. A large amount of neurosecretory material appeared in dorsomedial neurosecretory neurons in larvae fed diets with 100 and 250 microg Cd. Differences in larval brain protein profiles in the region of molecular mass ranges (Mr) of 98 kDa, 46 kDa and 3.4-6.1 kDa were identified in the experimental groups.

Biochemical analyses can point to toxicant presence before its effects can be detected at higher organizational levels. We investigated responses of larval mass and alkaline phosphatase (ALP) to different cadmium treatments in 4th instar gypsy moth larvae from 20 full-sib families. Changes in trait values and trait plasticities as well as their variation were monitored after acute and chronic exposure or recovery from two cadmium concentrations (Cd(1)=10microg and Cd(2)=30microg Cd/g dry food). Larval mass only decreased, without returning to the control level at recovery stage following chronic cadmium challenge. Acute stress did not change trait value but increased genetic variance of larval mass. Significant ALP activity changes, sensitivity of isozyme patterns (Mr of 60, 64, and 85kDa) and increased variation in ALP plasticity during acute exposure to cadmium point to its possible aplication as an exposure biomarker.

Searching for novel molecular biomarkers, we investigated cadmium effects on the ratio of specific activities of lysosomal and total acid phosphatases (ACPLys/ACPTot) in 4th instar gypsy moth larvae. After acute and chronic exposure to 10 and 30 μg Cd/g dry food, as well as after recovery from both concentrations, the trait values, plasticity, variability and genetic correlations were evaluated. The ACPLys/ACPTot ratio decreased during acute and chronic effects of both concentrations. Inhibition during long-term cadmium exposure was irreversible. Indices of phenotypic plasticity for ACPLys/ACPTot ratio were positive for all cadmium treatments. The variability of plasticity was higher after recovery from 10 μg Cd/g dry food, compared to recovery from 30 μg Cd/g dry food. A significant correlation coefficient was calculated between shortterm cadmium treatments. Significant changes in the ACPLys activity fraction during all treatments indicate the examined trait (ACPLys/ACPTot) could be used as a pollution exposure biomarker.

Gypsy moth 4th instar caterpillars were fed for 3 days with an artificial diet supplemented with increasing cadmium (Cd) concentrations (0, 10, 30, 100 and 250 μg⁄g of dry food weight). Changes in the morphometric characteristics of A1’ dorso-medial and L2 dorso-lateral neurosecretory neurons (nsn) were analyzed. In the A1’ nsn, Cd supplements led to an enhanced nuclear size, except in the group treated with 250 μg Cd⁄g in the form of dry food. The size of L2 type nsn was increased in the groups provided with 30 and 100 μg Cd⁄g, while no differences in the size of nuclei was detected in L2 neurons among the experimental groups.

A crucial enzyme in feeding and survival of Lymantria dispar (gypsy moth) caterpillars is β-glucosidase. Considering that this herbivorous species is one of the most damaging pests, a study of the enzyme that hydrolyzes plant polysaccharides is of invaluable significance. The knowledge of glucosidase alterations helps clarify our understanding of the insect pest feeding strategy in highly polluted environments. We sought to find out whether acute and chronic exposures to cadmium (10 and 30 mg Cd/g dry food), as well as the recovery from long-term treatment, influence the specific activity of midgut β-glucosidase. Cadmium ingestion did not alter enzyme activity compared to control, regardless of the treatment duration and metal concentration. The only statistically significant increase in enzyme activity was detected during the recovery after chronic treatment at 10 mg Cd/g dry food compared to the chronic cadmium effect at the same metal concentration. During chronic treatment at a lower cadmium concentration (10 mg Cd/g dry food), the phenotypic plasticity index was higher than at the recovery from the same concentration, while the variability of plasticity among these treatments was higher after recovery. Only one enzyme isoform was detected, which indicates its ability to hydrolyze several substrates. We concluded that this enzyme is probably essential for larval nutrition and survival in the presence of high amounts of cadmium in the food. [ABSTRACT FROM AUTHOR]

• The responses of gypsy moth larvae to Cd treatment depend on population origin. • Metallothionein level may be suitable for biomonitoring of Cd in the polluted areas. • Esterase activity is a potential biomarker of Cd exposure in the unpolluted forests. • Esterase isoform 3 could indicate an exposure to lower Cd concentrations. • IBR is a promising biomonitoring tool regardless of the exposure history. Long-term exposure of invertebrate populations to pollution might result in adaptations that must be taken into account when evaluating physiological parameters as biomarkers of contamination. In the present study we investigated whether previous exposure of gypsy moth, Lymantria dispar L. (Lepidoptera), population to pollution affects the sensitivity of larvae to cadmium treatment under laboratory conditions. Our goals in the current study were to compare metallothionein (MT) level and non-specific esterase activity including isoform expression in the midgut and fitness-related traits between caterpillars originating from polluted and unpolluted localities after chronic oral intake (50 and 100 µg Cd/g dry food). Besides being individually assessed as biomarkers of cadmium contamination, the responses of above parameters related to the midgut (MT concentration, specific activity of esterases and midgut mass) were summarized into the integrated biomarker response (IBR). Upon cadmium exposure, MT concentration increased significantly only in larvae originating from the polluted site, whereas non-specific esterase activity decreased in caterpillars from the unpolluted forest. We noticed similar patterns of esterase isoforms in both populations, including isoform number three that appeared only after exposure to the lower cadmium concentration. Midgut mass was reduced only in the group from the unpolluted locality exposed to the higher cadmium concentration. IBR index increased in a concentration-dependent manner in both populations, reflecting higher sensitivity of larvae from the unpolluted locality to the metal. Neither cadmium exposure nor population origin influenced larval survival. While MT concentration could be applied as a biomarker of cadmium exposure in populations that have been previously exposed to pollution for a longer time, non-specific esterase activity could indicate metal presence at unpolluted locations. IBR index and expression of esterase isoform number three could be used universally, regardless of the exposure history, except the latter might be limited to lower cadmium concentrations. [ABSTRACT FROM AUTHOR]

Long-term exposure of populations to pollution may result in enhanced ability to cope with environmental stress. To compare the responses of two Lymantria dispar populations living in unpolluted and polluted forests (UP and PP, respectively), we chronically exposed larvae to cadmium at concentrations of 50 and 100 μg Cd/g dry food (Cd1 and Cd2, respectively). We examined cadmium accumulation in the midgut and hemolymph, activities of superoxide dismutase (SOD), catalase (CAT), and alkaline phosphatases (ALP) in the midgut, as well as Hsp70 protein expression in the midgut, hemolymph, and brain and evaluated these parameters as biomarkers of cadmium contamination. Larvae from PP, fed a control diet, showed higher activity of SOD and increased Hsp70 expression compared with larvae from UP. Excessive amounts of Cd were accumulated in the midgut of all Cd-fed larvae, whereas Cd content in the hemolymph was elevated only in larvae from PP after Cd2 treatment. In larvae from UP, Cd2 treatment decreased the activity of CAT and induced the expression of Hsp70 in the midgut and hemolymph. In larvae from PP, exposure to both Cd concentrations strongly attenuated SOD and CAT activities, while Hsp70 expression was not induced in any organ/tissue. Cd did not affect ALP activity in either population. Midgut Cd content proved to be a suitable indicator of Cd contamination for both polluted and unpolluted habitats. [Display omitted] • Excessive amounts of Cd were accumulated in the midgut of all Cd-fed larvae. • Cd strongly attenuated SOD and CAT activities in larvae from the polluted site. • The activity of ALP was not affected in either population after Cd exposure. • Hsp70 expression was induced in Cd-fed larvae from the unpolluted locality. • Cd content, SOD and CAT activities, Hsp70 expression can be used for Cd biomonitoring. [ABSTRACT FROM AUTHOR]