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Cadmium pollution is becoming an increasing problem, especially in parts of the world that have developed industries. To consider the potentially harmful effects of cadmium, we need to examine changes at all different levels of biological organization. The main goal of this study was to detect a possible change in the percentage of midgut mass relative to larval mass (PMM) and determine the plasticity of this trait and the correlations between midgut enzymes and PMM under stress conditions. Fourth-instar larvae were exposed to acute and chronic effects of two cadmium concentrations, 10 and 30 μg Cd/g dry food, as well as a threeday recovery from chronic treatments. PMM is also an indirect indicator of food consumption and was found to be significantly reduced compared to control in both acute effects and chronic treatment at 30 μg and its three-day recovery. The PMM reduction during acute treatments is a consequence of cadmium action, while in chronic treatment, the genetic factor (egg mass) plays a crucial role in the change of PMM. According to the index of plasticity, distinct phenotypes were not produced. Significant correlations were shown between PMM and trypsin (Tryp) and leucine aminopeptidases (LAP) at acute and chronic treatment with higher cadmium concentrations, while significant correlations between proteases and PMM were detected at lower metal concentrations (Acute10 and Chronic10 and 30 μg Cd/g dry food). In contrast to chronic treatment, egg masses respond more uniformly by reducing PMM during the short-term effect of cadmium. Finally, we can conclude that, as an addition to biochemical and molecular research, PMM can be used for studying the cadmium effects to gain a better insight into the state of the organism under stress conditions. [ABSTRACT FROM AUTHOR]

Plant vegetation accumulates polycyclic aromatic hydrocarbons (PAHs) among which benzo[a]pyrene (B[a]P) is recognized as being very toxic, including cancerogenic. Lymantria dispar L. larvae are sensitive to changes in the environment, providing potential signs of pollutant presence. We examined the chronic effects of two concentrations of B[a] P on the activity of carboxylesterase (CaE), acetylcholinesterase (AChE) and heat shock protein 70 (Hsp70) levels in the brain tissue of two populations of L. dispar larvae, originating from unpolluted and polluted habitats. We found that the relative growth rate was significantly lower in both populations and that only larvae from polluted forests were sensitive to low B[a]P concentrations, exhibiting a significant increase in brain tissue CaE activity and Hsp70 concentration. AChE activity showed no changes in response to B[a]P exposure in either population. Examined biochemical parameters indicate that their sensitivity to chronic treatment with B[a]P was highly dependent on the pre-exposure history of L. dispar larvae, suggesting that they could be promising biomarkers of B[a]P and PAH pollution in forest ecosystems. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173027]

Gypsy moth caterpillars were exposed to an increased rearing temperature of 35ºC 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 treatment, suggesting intensive synthesis and secretory activity in L1 nsn. Caterpillars reared on an artificial substrate with the addition of high concentrations of tannic acid (TA) showed a decreased number of nsn, increased cell size and decreased size of their nuclei. The reduction in the relative density of the cytoplasm led us to conclude that this treatment induced a high synthetic activity of L1 nsn. [Projekat Ministarstva nauke Republike Srbije, br. 173027]

Lymantria dispar, a polyphagous insect pest, copes with a wide variety of host-specific allelochemicals. Glutathione S-transferases (GST) are important for catalyzing detoxification in L. dispar. Larval mortality, GST activity in midgut tissue and mass of L. dispar with different trophic adaptations (originating from two forests with a suitable host, Quercus robur, and an unsuitable host, Robinia pseudoacacia), differed after feeding on quercetin supplemented diets (2% or 5% w/w). Quercetin inhibited GST most potently in oak forest larvae that were less adapted to flavonoids in their diet. The larvicidal effect of quercetin on L. dispar larvae depended on the host-use history. We believe this is important in strategies for sustainable control of insect pests. [Projekat Ministarstva nauke Republike Srbije, br. 173027]

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.

The effects of allelochemical stress on genetic variations in the specific activities of gypsy moth digestive enzymes (trypsin and leucine aminopeptidase) and relative midgut mass (indirect measure of food consumption), as well as variability in their plasticity, were investigated in fifth instar gypsy moths originating from two populations with different trophic adaptations (oak and locust-tree forests). Thirty-two full-sib families from the Quercus population and twenty-six full-sib families from the Robinia population were reared on an artificial diet with or without supplementation with tannic acid. Between population differences were observed as higher average specific activity of trypsin and relative midgut mass in larvae from the Robinia population. Significant broad-sense heritabilities were observed for the specific activity of trypsin in the control state, and for specific activity of leucine aminopeptidase in a stressful environment. Significantly lower heritability for relative midgut mass was recorded in larvae from the Robinia population reared under stressful conditions. Significant variability of trypsin plasticity in larvae from both populations and significant variability of leucine aminopeptidase plasticity in larvae from the Robinia population point to the potential for the evolution of enzyme adaptive plastic responses to the presence of stressor. Non-significant across-environment genetic correlations do not represent a constraint for the evolution of enzyme plasticity. [Projekat Ministarstva nauke Republike Srbije, br. 173027]

In gypsy moth caterpillars exposed to a temperature of 35°C (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 23°C), 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.

The influence of allelochemical stress and population origin on the patterns of phenotypic and genetic correlations among life history traits and digestive enzyme activities were investigated in larvae of the gypsy moth (Lymantria dispar L.; Lepidoptera: Lymantriidae). Thirty-two full-sib families from oak (suitable host plant, Quercus population), and twenty-six full-sib families from locust-tree (unsuitable host plant, Robinia population) forests were reared on an artificial diet, with or without a 5% tannic acid supplement. Comparison of correlation matrices revealed significant similarity between the two populations in the structure of phenotypic and genetic correlations of life history traits and of digestive enzyme activities. The patterns of correlations of the examined traits, within each of the two locally adapted populations and in the presence of allelochemical stress, remained stabile despite the different selection pressures that mold these traits. [Projekat Ministarstva nauke Republike Srbije, br. 173027]

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, α- and β-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 α- and β-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.

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. [Acknowledgments. This study was supported by the Serbian Ministry of Education and Science (Grant No. 173027).]

The number, morphometric parameters and amount of aminergic neurosecretory product of protocerebral A2 neurosecretory neurons were investigated in the fifth instar of Lymantria dispar caterpillars, following a suitable or unsuitable trophic regime. Caterpillars originated from two populations (Quercus rubra or Robinia pseudoacacia forest) and were differently adapted to trophic stress, i.e. feeding on locust tree leaves - unsuitable host plant. The number of neurosecretory neurons was higher in the caterpillars originated from Robinia population than in Quercus population, regardless of feeding. A2 neurosecretory neurons, nuclei and their nucleoli were larger in caterpillars fed with unsuitable leaves in both populations. There was more aminergic product in the A2 neurosecretory neurons of the caterpillars fed with unsuitable leaves independently of population origin.

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.

In this study we investigated the effects of tannic acid on fitness-related traits in gypsy moth larvae from two differently adapted populations. Thirty two full-sib families from oak (suitable host plant, Quercus population), and twenty six full-sib families from locust-tree (unsuitable host plant, Robinia population) forests were reared on artificial diets with or without a 5% tannic acid supplement. Tannic acid shortened the duration of larval development till the 4th instar in the Robinia population, and decreased the mass of larvae from both Quercus and Robinia populations. Local adaptation was not recorded for any of the examined traits. In general, regardless of population origin, genetic variation (broad-sense heritability) was higher in larvae fed on the diet containing tannic acid than in larvae fed on the control diet. Variability of phenotypic plasticity was significant for the examined traits, except for larval duration traits in Quercus larvae, pointing to the potential of gypsy moth larvae from both populations for the evolution of adaptive plastic responses to new environmental conditions and the presence of stressors. Genetic correlations between the environments were positive and significantly different from “one” and accordingly, do not represent constraint for the evolution of plasticity.

The morphometric changes (size of neurons and their nuclei) of protocerebral dorsomedial A1’ and dorsolateral L2’ neurosecretory neurons were analyzed in Lymantria dispar larvae after exposure to strong static (SMF, 235 mT) and extremely low frequency magnetic fields (ELF MF, 2 mT). Increase in the size of A1’ neurons and their nuclei were observed after acute exposure to SMF. Decrease in the size of these neurons and their nuclei was observed after exposure to ELF MF. The size of L2’ neurons and their nuclei tend to decrease after exposure to SMF and ELF MF. The quantification of protein bands within the Mr range corresponding to the large form of the prothoracicotropic neurohormone indicates that the amount of protein decreased after exposure to both types of magnetic fields.

Morphometric changes of A1, A1' and A2 protocerebral dorsomedial neurosecretory neurons, total brain protein content and brain protein profiles were analyzed in 4th instar Lymantria dispar larvae under elevated rearing density, i.e. under intense stress when 5 larvae were kept in a petri dish (V = 80 ml), less intense stress when 5 larvae were kept in a plastic cup (V = 300 ml). In the control samples the larvae were reared in isolated conditions. Protein pattern changes in the brain were observed. Proteins with the following molecular masses: 30, 14, 10 and 3.4-2.5 kD were detected in the experimental groups. The size and cytological characteristics of protocerebral dorsomedial neurosecretory neurons were changed under elevated rearing density.

The effects of different temperatures (23°C and 8°C) on activity of corpora allata (CA) and dorsolateral (L1, L2) protocerebral neurosecretory neurons were investigated in Morimus funereus Mulsant (1863) larvae collected from a natural population during March. Activity of CA was revealed by monitoring of CA volume and cell number. Increase of CA volume after two day exposure to both temperatures was shown to be the result of increase in cell number. Activity of CA was higher at 23°C than 8°C. Activity of L1 and L2 neurosecretory neurons was inhibited at both temperatures. Neurosecretory neurons were more sensitive to temperature of 23°C than 8°C. It can be supposed that dorsolateral neurosecretory neurons synthesize neurohormones that affect CA activity, depending on environmental temperature.

The response of starved Morimus funereus larvae refed with an artificial diet (Galford, 1967) was examined in the present paper. Larvae were offered diets varying in protein quality (soya protein, casein, and gelatin). Refeeding with the Galford diet (G I) and two modifications of it in which soya protein was supplemented with casein (G II) or gelatin (G III) led to an increase of body mass. Different protein quality in the nutritive substrate influenced both the quantity and quality of midgut and brain proteins.