Dragana Matic | University of Belgrade (original) (raw)

Papers by Dragana Matic

Archives of Biological Sciences, 2013

In gypsy moth caterpillars exposed to a temperature of 35ºC (for 1, 12 and 24 h and caterpillars ... more In gypsy moth caterpillars exposed to a temperature of 35ºC (for 1, 12 and 24 h and caterpillars that were exposed to elevated temperature for 12 h and were allowed to recover for 12 h at 23ºC), changes in the brain protein profiles and morphometric characteristics of A1' medial and L2 lateral protocerebral neurosecretory neurons were analyzed. In all groups, protein bands with a molecular mass corresponding to that of members of heat-shock protein families were detected, indicating that acute exposure to this temperature likely induced the synthesis of HSP. Increased morphometric parameters of A1' neurons and the large amount of neurosecretory material in the neuron body implicate that the temperature of 35˚C is not in the temperature range that exerts stimulatory effects on growth and survival. Changes in the morphometric characteristics of L2 neurosecretory neurons from the lateral part of the protocerebrum, and retention of neurosecretory material in their cytoplasm indicate a low level of secretion.

Environmental Science and Pollution Research, Aug 9, 2017

The effects of different concentrations of benzo(a)pyrene (BaP) (2, 10, 20, 100, 200, and 2000 ng... more 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.

Clinical Neurophysiology, 2009

range. We used Student t-test for statistical analysis. Results: In ET patients we recordedM-pote... more range. We used Student t-test for statistical analysis. Results: In ET patients we recordedM-potential amplitude values of NP (6.77 ± 1.31 mV), which were statistically significantly lower than in CG (8.83 ± 1.64 mV). Other electrophysiological parameters values were not statistically different (p > 0.05) in two groups. Conclusions: ET may have some influence on single ENG parameter values, but most of ENG parameters in patients with ET are in normal ranges. Reduction of NP M-potential amplitude value in patients with ET may be associated with change of motor units function during tremor, chronic fatigue of muscles and other factors.

Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2017

Bioaccumulation and excretion of heavy metals in insects is only partially clarified. We have inv... more Bioaccumulation and excretion of heavy metals in insects is only partially clarified. We have investigated cadmium accumulation in the feces, head and integument of Lymantria dispar (Lepidoptera: Lymantriidae) larvae exposed to chronic dietary intake of cadmium. The aim of the experiment was to establish modalities of metal accumulation, primarily cadmium, as well as changes in fitness traits in two insect populations receiving 50 or 100 μg Cd/g dry food. The egg-masses originated from two localities: a protected nature reserve (unpolluted population) and an area near a busy highway (polluted population) in Serbia. At both added dietary levels cadmium concentration was highest in feces. Small alterations in metal concentrations after both cadmium treatments were detected in the integument. It was established that irrespective of population origin, the modality of decline of larval mass and relative growth rate (RGR) was similar. Concentrations of Cu and Zn in the integument were approximately the same regardless of dietary cadmium intake. However, cadmium accumulation in the examined tissues, as well as variability of fitness traits depended on population origin and cadmium concentration. In larvae from both populations not given contaminated food the head was the organ with the greatest accumulation of trace elements. Our results reveal how invasive phytophagous insects cope with high metal concentrations in their food by body mass (RGR) reduction and energy allocation towards processes that enable accumulation of cadmium and other trace elements in different tissues.

Environmental Pollution, 2016

The current study extends our previous findings concerning the sensitivity of Lymantria dispar la... more 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.

Chemosphere, 2016

Polycyclic aromatic hydrocarbons (PAHs) may affect biochemical and physiological processes in liv... more Polycyclic aromatic hydrocarbons (PAHs) may affect biochemical and physiological processes in living organisms, thus impairing fitness related traits and influencing their populations. This imposes the need for providing early-warning signals of pollution. Our study aimed to examine changes in the activity of acetylcholinesterase (AChE) and the concentration of heat shock proteins (Hsp70) in homogenates of brain tissues of fifth instar gypsy moth (Lymantria dispar L.) larvae, exposed to the ubiquitous PAH, fluoranthene, supplemented to the rearing diet. Significantly increased activity of AChE in larvae fed on the diets with high fluoranthene concentrations suggests the necessity for elucidation of the role of AChE in these insects when exposed to PAH pollution. Significant induction of Hsp70 in gypsy moth larvae reared on the diets containing low fluoranthene concentrations, indicate that changes in the level of Hsp70 might be useful as an indicator of pollution in this widespread forest species.

Environmental Toxicology and Pharmacology, 2016

Activity of glutathione S-transferase (GST) in midgut of gypsy moth caterpillars exposed to 10 an... more 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.

TURKISH JOURNAL OF BIOLOGY, 2015

The aim of the study was to examine changes in Cu-Zn superoxide dismutase (SOD), catalase (CAT), ... more The aim of the study was to examine changes in Cu-Zn superoxide dismutase (SOD), catalase (CAT), their gel electrophoresis profiles, glutathione reductase (GR) activity, amount of glutathione (GSH), and the activity of glutathione S-transferase (GST)-phase II biotransformation enzyme in the midgut tissue of gypsy moth (Lymantria dispar L. (Lepidoptera: Lymantriidae)) larvae after ghrelin treatment. Four subpicomolar injections of ghrelin (0.3 pmol) or physiological saline (control) were applied every 24 h. The SOD, CAT, GR, GST activity, and amount of GSH were higher in the ghrelin-treated group than in the control. Electrophoresis gel bands of SOD and CAT had higher area and density in the treated group. The effects of ghrelin on the antioxidative defense and GST activity in insects were detected for the first time. The results provided evidence for possible application of insects as simple model systems in future studies of the role of ghrelin in the antioxidative protection of complex organisms.

Archives of Biological Sciences, 2015

Gypsy moth caterpillars were exposed to an increased rearing temperature of 35?C and diet, supple... more Gypsy moth caterpillars were exposed to an increased rearing temperature of 35?C and diet, supplemented with Cd, a heavy metal pollutant, and tannic acid, a plant secondary metabolite. After 3 days? exposure to stressors, changes in the number, morphometric parameters of L1 neurosecretory neurons (nsn) (sizes of the nsn and their nuclei), and the quantity of neurosecretory material in the cytoplasm of the neurons were estimated. Acute exposure to the high temperature of 35?C induced increases in the number of L1 nsn, their size and the size of their nuclei with prolonged exposure time. After acute exposure to different Cd concentrations, the number of L1 nsn was reduced, their size increased and the size of their nuclei decreased. Together with the enhanced relative density of the cytoplasm, our results point to an intensive synthesis and retention of neurosecretory material in the neurons. The relative density of the neurosecretory material in the cytoplasm increased at the thermal...

Journal of Environmental Science and Health, Part B, 2015

We investigated the effects of acute and chronic treatments with cadmium at 10 μg Cd/g dry food a... more 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.

Biologia, 2014

Changes in the number and morphometric parameters of A1 neurosecretory neurons (nsn) were analyze... more Changes in the number and morphometric parameters of A1 neurosecretory neurons (nsn) were analyzed in Lymantria dispar 4th instar caterpillars, exposed for 3 days to different stressors: cadmium, high temperature and tannic acid. The relative cytoplasm density of A1 nsn was also estimated. Caterpillars reared on a diet supplemented with cadmium exhibited increased size of A1 nuclei (10 and 250 μg Cd per g of dry food weight), increased number of nucleolii in nuclei and raised relative cytoplasm density in all experimental groups. Cadmium obviously induces intensive synthetic activity in A1 nsn. The second stressor was high environmental temperature of 35°C. Decrease of all analyzed morphometric parameters suggests that acute exposure of 4th instar caterpillars to 35°C, as well as 12 h recovery at optimal temperature of 23°C, reduced the activity of A1 nsn. Tannic acid was added to the artificial diet in the following concentrations: 1%, 2.5% and 5%. All estimated morphological param...

TURKISH JOURNAL OF BIOLOGY, 2014

The release of neurosecretory material from A2 neurosecretory neurons (NSNs) was stimulated in Ly... more The release of neurosecretory material from A2 neurosecretory neurons (NSNs) was stimulated in Lymantria dispar fourth instar caterpillars exposed to a temperature of 35 °C for 1, 12, and 24 h, as well as those allowed recover after exposure (12 h at 35 °C, then 12 h at 23 °C). The levels of 2 protein forms with the same molecular mass as bombyxin (3-4 and 4-5 kDa) increased with prolonged exposure to 35 °C. The second band was present only in the groups exposed to this stressor. There was intensified synthetic activity and a low level of secretion in L2' NSNs after exposure to 35 °C. We previously found these NSNs to be immunopositive for prothoracicotropic neurohormone. After this stress, densitometric analysis revealed a decreased amount of the 11-12 kDa isoform (present in the control group). The new isoform (13-15 kDa), expressed after exposure of the insects to a high temperature, increased in amount with prolonged exposure and after recovery at 23 °C. Short-term exposure of caterpillars to high temperatures (35 °C) is a stressor and activates carbohydrate metabolism, while PTTH immunopositive NSNs are secretory-inactive during acute thermal stress regimes.

Peptides, 2012

Ghrelin is a 28-amino acid peptide that has significant effects on appetite and growth in humans ... more Ghrelin is a 28-amino acid peptide that has significant effects on appetite and growth in humans and animals. The aim of this study was to examine 4th instar larvae of the pest insect Lymantria dispar L. after ghrelin treatment. Parameters included changes in nutritional indices (efficiency of conversion of ingested food, efficiency of conversion of digested food, approximate digestibility); midgut and fat body mass; total proteases, trypsin and leucine aminopeptidase activities in the midgut; number, height and width of columnar and goblet cells and their nuclei in the midgut epithelium and detection of ghrelin-like immunoreactivity in the midgut tissue. Four subpicomolar injections of ghrelin (0.3pmol) or physiological saline (control) were applied every 24h. The nutritional indices were higher in the ghrelin treated than in the control group. Ghrelin treatment was also associated with elevation of midgut mass, induced digestive enzyme activities, increased fat body mass and morphometric changes in columnar and goblet cells. This is the first report of the presence of ghrelin-like hormone in endocrine cells of an insect midgut. Such information provides additional evidence for application of this relatively simple model system in the future studies of the mechanisms underlying of digestion and energy balance in more complex organisms.

Environmental Science and Pollution Research, 2012

Heavy metals damage the structure, chemistry, and function of cells, including enzyme systems ins... more 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.

Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2014

Cadmium uptake takes place mainly through food. Lymantria dispar larvae were exposed to dietary c... more Cadmium uptake takes place mainly through food. Lymantria dispar larvae were exposed to dietary cadmium in concentrations of 10 and 30 μg 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 30 μg Cd/dry food with no recovery on removal of cadmium for 3 days. 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.

Archives of Biological Sciences, 2011

Gypsy moth 4 th instar caterpillars were fed for 3 days with an artificial diet supplemented with... more Gypsy moth 4 th 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.

Archives of Biological Sciences, 2012

Lymantria dispar caterpillars were reared under two different rearing densities for the first thr... more Lymantria dispar caterpillars were reared under two different rearing densities for the first three days of the 4th larval instar: 5 larvae that were kept in a Petri dish (V = 80 ml) belonged to the intense stress (D1 group); 5 larvae that were kept in a plastic cup (V = 300ml) belonged to the group exposed to less intense stress (D2 group). In the control group, single larvae were reared in a Petri dish. Morphometric changes in L1, L2 and L2? dorsolateral neurosecretory neurons (nsn) were analyzed. After keeping 5 larvae in a Petri dish, the size of L2 neurosecretory neurons (nsn) significantly increased. Rearing 5 larvae in a plastic cup significantly increased the size of L1 nsn nuclei and the number of L2?nsn. A decrease in relative band densities in the region of molecular masses (11-15 kD) that correspond to prothoracicotropic hormones in the gypsy moth was observed in the electrophoretic profiles that were obtained after both treatments in comparison to the control group.

Archives of Biological Sciences, 2012

Ghrelin, along with several other hormones, has significant effects on appetite and growth in hum... more Ghrelin, along with several other hormones, has significant effects on appetite and growth in humans and animals. The aim of our study was to examine changes in relative growth rates, ?- and ?-glucosidase activities and endocrine cell size in the midgut of 4th instar caterpillars of the pest insect Lymantria dispar L. after ghrelin treatment. Four subpicomolar injections of ghrelin (0.3 pmol) or physiological saline were applied every 24 h to two separate groups of fifteen caterpillars. Repeated administration of ghrelin in subpicomolar doses elevated the relative growth rate, induced ?- and ?-glucosidase activities and increased the size of endocrine cells. The results are the first data about ghrelin effects on relative growth rate, digestive enzyme activities and midgut endocrine cells in insects. This information supports the use of this relatively simple model system in future studies of mechanisms underlying digestion in complex organisms.

Brain Research, 1997

Introduction As a gradogenic polyphagous and invasive species, the gypsy moth (Lymantria dispar L... more Introduction As a gradogenic polyphagous and invasive species, the gypsy moth (Lymantria dispar L.) encounters different environmental challenges. Considering that its host range is estimated at more than 500 plant species within 73 families (Liebhold et al., 1995), the gypsy moth is characterized by a variety of adaptations that ensure survival and reproduction in heterogeneous and stressful environments. Problems with gypsy moth overcrowding are not solved yet (Demir et al., 2009, 2012). Plants from the genus Quercus are accepted as the most suitable food and the most susceptible to L. dispar population eruptions (Maksimović, 1987; Liebhold et al., 1995). In the Republic of Serbia, large areas of forest and orchards were attacked (171,914 ha) by gypsy moths in 2012 according to the State Enterprise for Forest Management of Serbia (http:// www.srbijasume.rs/). Other parts of Europe are also at risk of gypsy moth invasion and degradation of forest ecosystems. This pest insect is one of the most serious insect defoliators of North American forests and urban landscapes according to the US Forest Service (http:// www.fs.fed.us/). Gypsy moth defoliation results in loss of growth, mortality of oak species, and shifts in species composition in mixed-oak forests (McGraw et al., 1990). Young oak leaves contain low amounts of flavonoids in spring when L. dispar attacks them (Salminen et al., 2004). A high population density of gypsy moths induces changes in leaf chemistry during defoliation, i.e. flavonoid content increases, leading to suppression of the insect immune response, and their resistance to viruses is lowered (Martemyanov et al., 2012). One of the very rare species of plant that is an unsuitable host for gypsy moths is the locust tree, Robinia pseudoacacia (Barbosa and Krischik, 1987). A nonfamiliar host plant for L. dispar, R. pseudoacacia is a widespread species in its native habitat in southeastern North America. It was introduced to Europe in 1601 (Chapman, 1935). Today, it has spread throughout western, central, eastern, and southern Europe and has become a major invasive species with a significant impact on native plant communities. Despite the large number of polyphagous species of Lepidoptera and the long period after the introduction of R. pseudoacacia to Europe, so far only a few native lepidopteran species have adapted to it (Kulfan, 2012). The unfavorable effects of locust leaves on gypsy moth larvae can be attributed to the presence of phenolic defensive compounds (Barbosa Abstract: As a very invasive insect species, Lymantria dispar is adaptable and sensitive to a changing environment. In insects the neuroendocrine system first reacts to stress by production of prothoracicotropic neurohormones (PTTH) that control ecdysteroid synthesis (morphogenetic and stress hormones). In this article, we report changes in the L2' brain neurosecretory neurons that synthesize PTTH in L. dispar larvae after feeding on locust tree leaves (Robinia pseudoacacia), an unsuitable host plant. Groups of larvae (n = 20 per experimental group) were offered this in comparison with oak leaves (Quercus robur), a suitable control diet, for 3 days after molting into the fourth instar. L2' neurons and their nuclei were enlarged and the amount of neurosecretory product in the cytoplasm was increased (15.5%) after consumption of locust tree leaves in comparison to the control. Furthermore, activities of the following antioxidative defense components were estimated: superoxide dismutase (SOD), catalase (CAT), and amount of glutathione in the midgut. Higher SOD activity (13.85 ± 0.9 U/mg prot.) and glutathione amount (0.56 ± 0.06 µMGSH/g tissue) but unchanged CAT activity was found in the midgut of larvae offered locust tree leaves when compared to the control.

Molecular and Cellular Biochemistry, 2010

The aim of this study was to investigate the dynamics of lipid peroxidation and the possible corr... more The aim of this study was to investigate the dynamics of lipid peroxidation and the possible correlation between lipid peroxidation in different brain regions and behavioral manifestations in lindane-induced seizures in rats. Male Wistar rats were divided into the following groups: 1. control, saline-treated group; 2. dimethylsulfoxide (DMSO)-treated group; 3. lindane-treated group (8 mg/kg), intraperitoneally. Animals were sacrificed 0.5 or 4 h after treatment and the malondialdehyde level and superoxide dismutase (SOD) activity were determined in various brain regions spectrophotometrically. Behavioral changes were classified according to the descriptive scale (0—no response, 1—head nodding, lower jaw twitching; 2—myoclonic body jerks, bilateral forelimb clonus with full rearing; 3—progression to generalized clonic convulsions followed by tonic extension of fore- and hind limbs and tail; 4—status epilepticus). A significant rise in the malondialdehyde level was detected in the cerebral cortex, hippocampus, and thalamus of lindane-treated animals 0.5 and 4 h after administration (P < 0.05). SOD activity (total and mitochondrial) was significantly decreased in the hippocampus and the cortex of lindane-treated animals at both time points (P < 0.05). An initial fall in SOD activity was detected in the thalamus 4 h after lindane administration (P < 0.05). A positive correlation between seizure severity and the malondialdehyde level was found in the hippocampus at both time points (P < 0.01). These results suggest that lipid peroxidation may contribute to the neurotoxic effects of lindane in early acute lindane intoxication and that behavioral manifestations correlate with lipid peroxidation in the hippocampus of lindane-treated rats.

Archives of Biological Sciences, 2013

In gypsy moth caterpillars exposed to a temperature of 35ºC (for 1, 12 and 24 h and caterpillars ... more In gypsy moth caterpillars exposed to a temperature of 35ºC (for 1, 12 and 24 h and caterpillars that were exposed to elevated temperature for 12 h and were allowed to recover for 12 h at 23ºC), changes in the brain protein profiles and morphometric characteristics of A1' medial and L2 lateral protocerebral neurosecretory neurons were analyzed. In all groups, protein bands with a molecular mass corresponding to that of members of heat-shock protein families were detected, indicating that acute exposure to this temperature likely induced the synthesis of HSP. Increased morphometric parameters of A1' neurons and the large amount of neurosecretory material in the neuron body implicate that the temperature of 35˚C is not in the temperature range that exerts stimulatory effects on growth and survival. Changes in the morphometric characteristics of L2 neurosecretory neurons from the lateral part of the protocerebrum, and retention of neurosecretory material in their cytoplasm indicate a low level of secretion.

Environmental Science and Pollution Research, Aug 9, 2017

The effects of different concentrations of benzo(a)pyrene (BaP) (2, 10, 20, 100, 200, and 2000 ng... more 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.

Clinical Neurophysiology, 2009

range. We used Student t-test for statistical analysis. Results: In ET patients we recordedM-pote... more range. We used Student t-test for statistical analysis. Results: In ET patients we recordedM-potential amplitude values of NP (6.77 ± 1.31 mV), which were statistically significantly lower than in CG (8.83 ± 1.64 mV). Other electrophysiological parameters values were not statistically different (p > 0.05) in two groups. Conclusions: ET may have some influence on single ENG parameter values, but most of ENG parameters in patients with ET are in normal ranges. Reduction of NP M-potential amplitude value in patients with ET may be associated with change of motor units function during tremor, chronic fatigue of muscles and other factors.

Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2017

Bioaccumulation and excretion of heavy metals in insects is only partially clarified. We have inv... more Bioaccumulation and excretion of heavy metals in insects is only partially clarified. We have investigated cadmium accumulation in the feces, head and integument of Lymantria dispar (Lepidoptera: Lymantriidae) larvae exposed to chronic dietary intake of cadmium. The aim of the experiment was to establish modalities of metal accumulation, primarily cadmium, as well as changes in fitness traits in two insect populations receiving 50 or 100 μg Cd/g dry food. The egg-masses originated from two localities: a protected nature reserve (unpolluted population) and an area near a busy highway (polluted population) in Serbia. At both added dietary levels cadmium concentration was highest in feces. Small alterations in metal concentrations after both cadmium treatments were detected in the integument. It was established that irrespective of population origin, the modality of decline of larval mass and relative growth rate (RGR) was similar. Concentrations of Cu and Zn in the integument were approximately the same regardless of dietary cadmium intake. However, cadmium accumulation in the examined tissues, as well as variability of fitness traits depended on population origin and cadmium concentration. In larvae from both populations not given contaminated food the head was the organ with the greatest accumulation of trace elements. Our results reveal how invasive phytophagous insects cope with high metal concentrations in their food by body mass (RGR) reduction and energy allocation towards processes that enable accumulation of cadmium and other trace elements in different tissues.

Environmental Pollution, 2016

The current study extends our previous findings concerning the sensitivity of Lymantria dispar la... more 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.

Chemosphere, 2016

Polycyclic aromatic hydrocarbons (PAHs) may affect biochemical and physiological processes in liv... more Polycyclic aromatic hydrocarbons (PAHs) may affect biochemical and physiological processes in living organisms, thus impairing fitness related traits and influencing their populations. This imposes the need for providing early-warning signals of pollution. Our study aimed to examine changes in the activity of acetylcholinesterase (AChE) and the concentration of heat shock proteins (Hsp70) in homogenates of brain tissues of fifth instar gypsy moth (Lymantria dispar L.) larvae, exposed to the ubiquitous PAH, fluoranthene, supplemented to the rearing diet. Significantly increased activity of AChE in larvae fed on the diets with high fluoranthene concentrations suggests the necessity for elucidation of the role of AChE in these insects when exposed to PAH pollution. Significant induction of Hsp70 in gypsy moth larvae reared on the diets containing low fluoranthene concentrations, indicate that changes in the level of Hsp70 might be useful as an indicator of pollution in this widespread forest species.

Environmental Toxicology and Pharmacology, 2016

Activity of glutathione S-transferase (GST) in midgut of gypsy moth caterpillars exposed to 10 an... more 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.

TURKISH JOURNAL OF BIOLOGY, 2015

The aim of the study was to examine changes in Cu-Zn superoxide dismutase (SOD), catalase (CAT), ... more The aim of the study was to examine changes in Cu-Zn superoxide dismutase (SOD), catalase (CAT), their gel electrophoresis profiles, glutathione reductase (GR) activity, amount of glutathione (GSH), and the activity of glutathione S-transferase (GST)-phase II biotransformation enzyme in the midgut tissue of gypsy moth (Lymantria dispar L. (Lepidoptera: Lymantriidae)) larvae after ghrelin treatment. Four subpicomolar injections of ghrelin (0.3 pmol) or physiological saline (control) were applied every 24 h. The SOD, CAT, GR, GST activity, and amount of GSH were higher in the ghrelin-treated group than in the control. Electrophoresis gel bands of SOD and CAT had higher area and density in the treated group. The effects of ghrelin on the antioxidative defense and GST activity in insects were detected for the first time. The results provided evidence for possible application of insects as simple model systems in future studies of the role of ghrelin in the antioxidative protection of complex organisms.

Archives of Biological Sciences, 2015

Gypsy moth caterpillars were exposed to an increased rearing temperature of 35?C and diet, supple... more Gypsy moth caterpillars were exposed to an increased rearing temperature of 35?C and diet, supplemented with Cd, a heavy metal pollutant, and tannic acid, a plant secondary metabolite. After 3 days? exposure to stressors, changes in the number, morphometric parameters of L1 neurosecretory neurons (nsn) (sizes of the nsn and their nuclei), and the quantity of neurosecretory material in the cytoplasm of the neurons were estimated. Acute exposure to the high temperature of 35?C induced increases in the number of L1 nsn, their size and the size of their nuclei with prolonged exposure time. After acute exposure to different Cd concentrations, the number of L1 nsn was reduced, their size increased and the size of their nuclei decreased. Together with the enhanced relative density of the cytoplasm, our results point to an intensive synthesis and retention of neurosecretory material in the neurons. The relative density of the neurosecretory material in the cytoplasm increased at the thermal...

Journal of Environmental Science and Health, Part B, 2015

We investigated the effects of acute and chronic treatments with cadmium at 10 μg Cd/g dry food a... more 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.

Biologia, 2014

Changes in the number and morphometric parameters of A1 neurosecretory neurons (nsn) were analyze... more Changes in the number and morphometric parameters of A1 neurosecretory neurons (nsn) were analyzed in Lymantria dispar 4th instar caterpillars, exposed for 3 days to different stressors: cadmium, high temperature and tannic acid. The relative cytoplasm density of A1 nsn was also estimated. Caterpillars reared on a diet supplemented with cadmium exhibited increased size of A1 nuclei (10 and 250 μg Cd per g of dry food weight), increased number of nucleolii in nuclei and raised relative cytoplasm density in all experimental groups. Cadmium obviously induces intensive synthetic activity in A1 nsn. The second stressor was high environmental temperature of 35°C. Decrease of all analyzed morphometric parameters suggests that acute exposure of 4th instar caterpillars to 35°C, as well as 12 h recovery at optimal temperature of 23°C, reduced the activity of A1 nsn. Tannic acid was added to the artificial diet in the following concentrations: 1%, 2.5% and 5%. All estimated morphological param...

TURKISH JOURNAL OF BIOLOGY, 2014

The release of neurosecretory material from A2 neurosecretory neurons (NSNs) was stimulated in Ly... more The release of neurosecretory material from A2 neurosecretory neurons (NSNs) was stimulated in Lymantria dispar fourth instar caterpillars exposed to a temperature of 35 °C for 1, 12, and 24 h, as well as those allowed recover after exposure (12 h at 35 °C, then 12 h at 23 °C). The levels of 2 protein forms with the same molecular mass as bombyxin (3-4 and 4-5 kDa) increased with prolonged exposure to 35 °C. The second band was present only in the groups exposed to this stressor. There was intensified synthetic activity and a low level of secretion in L2' NSNs after exposure to 35 °C. We previously found these NSNs to be immunopositive for prothoracicotropic neurohormone. After this stress, densitometric analysis revealed a decreased amount of the 11-12 kDa isoform (present in the control group). The new isoform (13-15 kDa), expressed after exposure of the insects to a high temperature, increased in amount with prolonged exposure and after recovery at 23 °C. Short-term exposure of caterpillars to high temperatures (35 °C) is a stressor and activates carbohydrate metabolism, while PTTH immunopositive NSNs are secretory-inactive during acute thermal stress regimes.

Peptides, 2012

Ghrelin is a 28-amino acid peptide that has significant effects on appetite and growth in humans ... more Ghrelin is a 28-amino acid peptide that has significant effects on appetite and growth in humans and animals. The aim of this study was to examine 4th instar larvae of the pest insect Lymantria dispar L. after ghrelin treatment. Parameters included changes in nutritional indices (efficiency of conversion of ingested food, efficiency of conversion of digested food, approximate digestibility); midgut and fat body mass; total proteases, trypsin and leucine aminopeptidase activities in the midgut; number, height and width of columnar and goblet cells and their nuclei in the midgut epithelium and detection of ghrelin-like immunoreactivity in the midgut tissue. Four subpicomolar injections of ghrelin (0.3pmol) or physiological saline (control) were applied every 24h. The nutritional indices were higher in the ghrelin treated than in the control group. Ghrelin treatment was also associated with elevation of midgut mass, induced digestive enzyme activities, increased fat body mass and morphometric changes in columnar and goblet cells. This is the first report of the presence of ghrelin-like hormone in endocrine cells of an insect midgut. Such information provides additional evidence for application of this relatively simple model system in the future studies of the mechanisms underlying of digestion and energy balance in more complex organisms.

Environmental Science and Pollution Research, 2012

Heavy metals damage the structure, chemistry, and function of cells, including enzyme systems ins... more 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.

Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2014

Cadmium uptake takes place mainly through food. Lymantria dispar larvae were exposed to dietary c... more Cadmium uptake takes place mainly through food. Lymantria dispar larvae were exposed to dietary cadmium in concentrations of 10 and 30 μg 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 30 μg Cd/dry food with no recovery on removal of cadmium for 3 days. 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.

Archives of Biological Sciences, 2011

Gypsy moth 4 th instar caterpillars were fed for 3 days with an artificial diet supplemented with... more Gypsy moth 4 th 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.

Archives of Biological Sciences, 2012

Lymantria dispar caterpillars were reared under two different rearing densities for the first thr... more Lymantria dispar caterpillars were reared under two different rearing densities for the first three days of the 4th larval instar: 5 larvae that were kept in a Petri dish (V = 80 ml) belonged to the intense stress (D1 group); 5 larvae that were kept in a plastic cup (V = 300ml) belonged to the group exposed to less intense stress (D2 group). In the control group, single larvae were reared in a Petri dish. Morphometric changes in L1, L2 and L2? dorsolateral neurosecretory neurons (nsn) were analyzed. After keeping 5 larvae in a Petri dish, the size of L2 neurosecretory neurons (nsn) significantly increased. Rearing 5 larvae in a plastic cup significantly increased the size of L1 nsn nuclei and the number of L2?nsn. A decrease in relative band densities in the region of molecular masses (11-15 kD) that correspond to prothoracicotropic hormones in the gypsy moth was observed in the electrophoretic profiles that were obtained after both treatments in comparison to the control group.

Archives of Biological Sciences, 2012

Ghrelin, along with several other hormones, has significant effects on appetite and growth in hum... more Ghrelin, along with several other hormones, has significant effects on appetite and growth in humans and animals. The aim of our study was to examine changes in relative growth rates, ?- and ?-glucosidase activities and endocrine cell size in the midgut of 4th instar caterpillars of the pest insect Lymantria dispar L. after ghrelin treatment. Four subpicomolar injections of ghrelin (0.3 pmol) or physiological saline were applied every 24 h to two separate groups of fifteen caterpillars. Repeated administration of ghrelin in subpicomolar doses elevated the relative growth rate, induced ?- and ?-glucosidase activities and increased the size of endocrine cells. The results are the first data about ghrelin effects on relative growth rate, digestive enzyme activities and midgut endocrine cells in insects. This information supports the use of this relatively simple model system in future studies of mechanisms underlying digestion in complex organisms.

Brain Research, 1997

Introduction As a gradogenic polyphagous and invasive species, the gypsy moth (Lymantria dispar L... more Introduction As a gradogenic polyphagous and invasive species, the gypsy moth (Lymantria dispar L.) encounters different environmental challenges. Considering that its host range is estimated at more than 500 plant species within 73 families (Liebhold et al., 1995), the gypsy moth is characterized by a variety of adaptations that ensure survival and reproduction in heterogeneous and stressful environments. Problems with gypsy moth overcrowding are not solved yet (Demir et al., 2009, 2012). Plants from the genus Quercus are accepted as the most suitable food and the most susceptible to L. dispar population eruptions (Maksimović, 1987; Liebhold et al., 1995). In the Republic of Serbia, large areas of forest and orchards were attacked (171,914 ha) by gypsy moths in 2012 according to the State Enterprise for Forest Management of Serbia (http:// www.srbijasume.rs/). Other parts of Europe are also at risk of gypsy moth invasion and degradation of forest ecosystems. This pest insect is one of the most serious insect defoliators of North American forests and urban landscapes according to the US Forest Service (http:// www.fs.fed.us/). Gypsy moth defoliation results in loss of growth, mortality of oak species, and shifts in species composition in mixed-oak forests (McGraw et al., 1990). Young oak leaves contain low amounts of flavonoids in spring when L. dispar attacks them (Salminen et al., 2004). A high population density of gypsy moths induces changes in leaf chemistry during defoliation, i.e. flavonoid content increases, leading to suppression of the insect immune response, and their resistance to viruses is lowered (Martemyanov et al., 2012). One of the very rare species of plant that is an unsuitable host for gypsy moths is the locust tree, Robinia pseudoacacia (Barbosa and Krischik, 1987). A nonfamiliar host plant for L. dispar, R. pseudoacacia is a widespread species in its native habitat in southeastern North America. It was introduced to Europe in 1601 (Chapman, 1935). Today, it has spread throughout western, central, eastern, and southern Europe and has become a major invasive species with a significant impact on native plant communities. Despite the large number of polyphagous species of Lepidoptera and the long period after the introduction of R. pseudoacacia to Europe, so far only a few native lepidopteran species have adapted to it (Kulfan, 2012). The unfavorable effects of locust leaves on gypsy moth larvae can be attributed to the presence of phenolic defensive compounds (Barbosa Abstract: As a very invasive insect species, Lymantria dispar is adaptable and sensitive to a changing environment. In insects the neuroendocrine system first reacts to stress by production of prothoracicotropic neurohormones (PTTH) that control ecdysteroid synthesis (morphogenetic and stress hormones). In this article, we report changes in the L2' brain neurosecretory neurons that synthesize PTTH in L. dispar larvae after feeding on locust tree leaves (Robinia pseudoacacia), an unsuitable host plant. Groups of larvae (n = 20 per experimental group) were offered this in comparison with oak leaves (Quercus robur), a suitable control diet, for 3 days after molting into the fourth instar. L2' neurons and their nuclei were enlarged and the amount of neurosecretory product in the cytoplasm was increased (15.5%) after consumption of locust tree leaves in comparison to the control. Furthermore, activities of the following antioxidative defense components were estimated: superoxide dismutase (SOD), catalase (CAT), and amount of glutathione in the midgut. Higher SOD activity (13.85 ± 0.9 U/mg prot.) and glutathione amount (0.56 ± 0.06 µMGSH/g tissue) but unchanged CAT activity was found in the midgut of larvae offered locust tree leaves when compared to the control.

Molecular and Cellular Biochemistry, 2010

The aim of this study was to investigate the dynamics of lipid peroxidation and the possible corr... more The aim of this study was to investigate the dynamics of lipid peroxidation and the possible correlation between lipid peroxidation in different brain regions and behavioral manifestations in lindane-induced seizures in rats. Male Wistar rats were divided into the following groups: 1. control, saline-treated group; 2. dimethylsulfoxide (DMSO)-treated group; 3. lindane-treated group (8 mg/kg), intraperitoneally. Animals were sacrificed 0.5 or 4 h after treatment and the malondialdehyde level and superoxide dismutase (SOD) activity were determined in various brain regions spectrophotometrically. Behavioral changes were classified according to the descriptive scale (0—no response, 1—head nodding, lower jaw twitching; 2—myoclonic body jerks, bilateral forelimb clonus with full rearing; 3—progression to generalized clonic convulsions followed by tonic extension of fore- and hind limbs and tail; 4—status epilepticus). A significant rise in the malondialdehyde level was detected in the cerebral cortex, hippocampus, and thalamus of lindane-treated animals 0.5 and 4 h after administration (P < 0.05). SOD activity (total and mitochondrial) was significantly decreased in the hippocampus and the cortex of lindane-treated animals at both time points (P < 0.05). An initial fall in SOD activity was detected in the thalamus 4 h after lindane administration (P < 0.05). A positive correlation between seizure severity and the malondialdehyde level was found in the hippocampus at both time points (P < 0.01). These results suggest that lipid peroxidation may contribute to the neurotoxic effects of lindane in early acute lindane intoxication and that behavioral manifestations correlate with lipid peroxidation in the hippocampus of lindane-treated rats.