Digestive enzyme activity and macromolecule content in the hemolymph of differentially adapted Lymantria dispar L. populations after short-term increases in ambient temperature.

Global, unpredictable temperature increases have strong effects on all organisms, especially insects. Elucidating the effects of short-term temperature increases on midgut digestive enzymes (α-glucosidase, lipase, trypsin, and leucine aminopeptidase - LAP) and metabolic macromolecules in the hemolymph (proteins, lipids, and trehalose) of phytophagous pest larvae of Lymantria dispar is important for general considerations of insect adaptation to a warming climate and potential pest control options. We also wanted to determine whether the different adaptations of L. dispar populations to environmental pollution might affect their ability to cope with heat stress using larvae from the undisturbed, Kosmaj forest and disturbed, Lipovica forest. Heat treatments at 28 °C increased α-glucosidase activity in both larval populations, inhibited LAP activity in larvae from the polluted forest, and had no significant effect on trypsin and lipase activities, regardless of larval origin. The concentration of proteins, lipids, and trehalose in the hemolymph of larvae from the disturbed forest increased, whereas the population from the undisturbed forest showed only an increase in proteins and lipids after the heat treatments. Larval mass was also increased in larvae from the undisturbed forest. Our results suggest a higher sensitivity of digestive enzymes and metabolism to short-term heat stress in L. dispar populations adapted to pollution in their forest habitat, although climate warming is not beneficial even for populations from unpolluted forests. The digestive and metabolic processes of L. dispar larvae are substantially affected by sublethal short-term increases in ambient temperature.

Effects of dietary fluoranthene on tissue-specific responses of carboxylesterases, acetylcholinesterase and heat shock protein 70 in two forest lepidopteran species.

In this study, responses of carboxylesterases, acetylcholinesterase, and stress protein Hsp70 were examined in the midgut and midgut tissue, and brain of fifth instar larvae of Lymantria dispar L. and Euproctis chrysorrhoea L. following chronic exposure to dietary fluoranthene. Specific carboxylesterase activity increased significantly in the midgut tissue of E. chrysorrhoea larvae treated with a lower fluoranthene concentration. The specific patterns of isoforms expression, recorded in larvae of both species, enable efficient carboxylesterase activity as a significant part of defense mechanisms. Increased Hsp70 concentration in the brain of L. dispar larvae points to a response to the proteotoxic effects of a lower fluoranthene concentration. Decreased Hsp70 in the brain of E. chrysorrhoea larvae in both treated groups can suggest induction of other mechanisms of defense. The results indicate the importance of the examined parameters in larvae of both species exposed to the pollutant, as well as their potential as biomarkers.

Biological effects of chronic exposure of Blaptica dubia (Blattodea: Blaberidae) nymphs to static and extremely low frequency magnetic fields.

In this paper, we analyzed the effects of chronic exposure (5 months) to static magnetic field (110 mT; SMF) and extremely low frequency magnetic field (ELF MF; 10 mT, 50 Hz) on Blaptica dubia nymphs. We have examined acetylcholinesterase (AChE) activity and heat shock protein 70 (HSP70) level, two sensitive biomarkers of stress in terrestrial insects. Relative growth rate (RGR), as a life history trait, was estimated. AChE activity was determined spectrophotometrically and HSP70 levels were quantified using indirect non-competitive ELISA and Western blotting. Calculated RGR was significantly changed upon exposure to both types of ambiental MFs. The effects of chronic exposure of B. dubia nymphs to SMF and ELF MF (50 Hz) were observed as decreased activity of AChE. The increased level of HSP70 was present only after exposure to SMF. The strength of ELF MF was most likely below the energy level needed to induce the expression of this stress protein. Different patterns of the expression of two HSP70 isoforms, where isoform 2 was sensitive only to SMF, are most likely a possibly switch - off in the expression of constitutive and/or inducible HSP70 isoforms.

Evaluation of oxidative stress biomarkers in the freshwater gammarid Gammarus dulensis exposed to trout farm outputs.

The current study reports a combined seasonal monitoring of the Crnica River watercourse using physico-chemical parameters and biomarkers measured in Gammarus dulensis with the aim of correlating the effects of pollution with the level of oxidative stress biomarkers in this amphipod. The pollution source is identified as a trout farm in the upper part of the Crnica River (Eastern Serbia). Physico-chemical water parameters and sediment trace element concentrations were measured in the Crnica River during three seasons. Three localities downstream [CR2, CR3, and CR4 (20 m, 400 m, and 1.3 km from the trout farm discharge, respectively)] were assessed in comparison with a reference location (CR1), 250 m upstream from the trout aquaculture in the Crnica River. Antioxidant biomarkers including the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione-S-transferase (GST) were determined in pooled samples of G. dulensis. Our findings showed statistically different antioxidant enzyme levels in gammarids from the first downstream locality (CR2) compared to those from other three localities. The results indicate that oxidative stress occurred to some extent despite the assumed antioxidant induction as a result of exposure to elevated levels of some elements and changed water parameters, the effects seeming in part to be mediated by suppression of SOD and CAT (during summer and autumn); and by suppression of GPX and GR (during autumn) at the CR2 locality. The activity of GST in all seasons was higher at the CR2 locality than at the other localities, suggesting the presence of some permanent xenobiotic next to the aquaculture output. Seasonal difference in the physical and chemical water parameters and sediment trace element concentrations were observed. Overall, our results indicate a locality- and season-dependent antioxidant enzymes response in G. dulensis, pointing at progressively reduced deleterious effect of trout farm outputs with movement further downstream.

Photonic structures improve radiative heat exchange of Rosalia alpina (Coleoptera: Cerambycidae).

The insect cuticle serves a multitude of purposes, including: mechanical and thermal protection, water-repelling, acoustic signal absorption and coloration. The influence of cuticular structures on infrared radiation exchange and thermal balance is still largely unexplored. Here we report on the micro- and nanostructured setae covering the elytra of the longicorn beetle Rosalia alpina (Linnaeus, 1758) (Coleoptera: Cerambycidae) that help the insect to survive in hot, summer environments. In the visible part of the spectrum, scale-like setae, covering the black patches of the elytra, efficiently absorb light due to the radiation trap effect. In the infrared part of the spectrum, setae of the whole elytra significantly contribute to the radiative heat exchange. From the biological point of view, insect elytra facilitate camouflage, enable rapid heating to the optimum body temperature and prevent overheating by emitting excess thermal energy.

Short- and long-term exposure to alternating magnetic field (50 Hz, 0.5 mT) affects rat pituitary ACTH cells: Stereological study.

The aim of the present study was to determine does extremely low frequency magnetic field (ELF-MF, 50 Hz, 0.5 mT) affect pituitary adrenocorticotroph (ACTH) cells in adult animals. We performed two series of experiments: (1) short-term exposure of 3-month-old rats to ELF-MF for 1 and 7 days, and (2) long-term exposure of rats to ELF-MF from their conception to 3 months of age. Stereological study was performed on immunolabeled pituitary ACTH cells. The total number and volume of ACTH cells, the volume of their nuclei and pituitary volume were measured. ELF-MF exposure for 1 day significantly decreased total number and volume of ACTH cells, the volume of their nuclei, as well as pituitary volume. ELF-MF exposure for 7 days significantly reduced only the volume of ACTH cells. Life-long exposure to ELF-MF induced decrease in the volume of ACTH cells and pituitary volume. We can conclude that the applied ELF-MF has a strong influence on morphometrical parameters of the pituitary ACTH cells and could be considered as a stressogenic factor.

Effects of the static and ELF magnetic fields on the neuronal population activity in Morimus funereus (Coleoptera, Cerambycidae) antennal lobe revealed by wavelet analysis.

To study the influence of a static magnetic field (SMF, 2 mT) and extremely low frequency magnetic field (ELF MF, 50 Hz, 2 mT) on the neuronal population activity, the experiments were performed on adult longhorn beetles Morimus funereus (Coleoptera, Cerambycidae). Based on a wavelet analysis of the local field potentials (LFPs), our study showed for the first time that the effects of prolonged and repeated exposure to the ELF MF on the LFPs were irreversible within investigated time frame. The relative wavelet energy (RWE) of 4-8 Hz frequency band was significantly increased after sine ELF MF (SnMF)/square ELF MF (SqMF) in comparison to the control value. The RWE of slower oscillations (1-2Hz) was significantly decreased after the repeated exposures to either SnMF or SqMF. The SqMF induced decreasing of the faster waves in the range of 64-128 Hz. However, we did not prove with presented methods that exposure to the SMF for 5 min produces any effects on the neuronal population activity. This study has proved the wavelet transform as a valuable tool for measuring the effects of SMF and ELF MF on the neuronal population activity in M. funereus antennal lobe.

Response of α-glucosidase in gypsy moth larvae to acute and chronic dietary cadmium.

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.

Responses of PTTH-producing neurosecretory neurons in Lymantria dispar caterpillars exposed to cadmium.

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.

Sourcing chitin from exoskeleton of Tenebrio molitor fed with polystyrene or plastic kitchen wrap.

In this work we have characterized and compared chitin sourced from exoskeleton of Tenebrio molitor larvae fed with polystyrene or plastic kitchen wrap combined with bran in the ratio 1: 1 with chitin sourced from larvae exoskeleton fed only with bran. Analysis of the frass by ATR-FTIR showed very similar spectra and confirmed degradation of the plastic feed components, while ATR-FTIR analysis of the exoskeleton verified the absence of any plastic residue. Deproteinization followed by demineralization produced 6.78-5.29 % chitin, showing that plastic (polystyrene or plastic kitchen wrap) in the larvae diet resulted in heavier insect exoskeleton, but yielded slightly less chitin, with the lowest value obtained for plastic kitchen wrap in the insect diet. The deacetylation degree of 98.17-98.61 % was determined from measured ATR-FTIR spectra. XRD analysis confirmed the presence of α-chitin with a crystallinity index of 66.5-62 % and crystallite size 4-5 nm. Thermogravimetric analysis showed similar degradation curves for all chitin samples, with two degradation steps. These results show that chitin sourced from exoskeleton of T. molitor larvae fed with plastic (polystyrene or plastic kitchen wrap) and contributing to significant biodegradation of major polluting materials can be a feasible and alternative source of chitin, further promoting a bio-circular economy.

Changes in the expression and current of the Na+/K+ pump in the snail nervous system after exposure to a static magnetic field.

Compelling evidence supports the use of a moderate static magnetic field (SMF) for therapeutic purposes. In order to provide insight into the mechanisms underlying SMF treatment, it is essential to examine the cellular responses elicited by therapeutically applied SMF, especially in the nervous system. The Na(+)/K(+) pump, by creating and maintaining the gradient of Na(+) and K(+) ions across the plasma membrane, regulates the physiological properties of neurons. In this study, we examined the expression of the Na(+)/K(+) pump in the isolated brain-subesophageal ganglion complex of the garden snail Helix pomatia, along with the immunoreactivity and current of the Na(+)/K(+) pump in isolated snail neurons after 15 min exposure to a moderate (10 mT) SMF. Western blot and immunofluorescence analysis revealed that 10 mT SMF did not significantly change the expression of the Na(+)/K(+) pump α-subunit in the snail brain and the neuronal cell body. However, our immunofluorescence data showed that SMF treatment induced a significant increase in the Na(+)/K(+) pump α-subunit expression in the neuronal plasma membrane area. This change in Na(+)/K(+) pump expression was reflected in pump activity as demonstrated by the pump current measurements. Whole-cell patch-clamp recordings from isolated snail neurons revealed that Na(+)/K(+) pump current density was significantly increased after the 10 mT SMF treatment. The SMF-induced increase was different in the two groups of control snail neurons, as defined by the pump current level. The results obtained could represent a physiologically important response of neurons to 10 mT SMF comparable in strength to therapeutic applications.

Effect of magnetic fields on antioxidative defense and fitness-related traits of Baculum extradentatum (insecta, phasmatodea).

This study aimed to determine the effect of magnetic fields on the antioxidative defense and fitness-related traits of Baculum extradentatum. Following exposure to magnetic fields, antioxidative defense (superoxide dismutase (SOD), catalase (CAT) activities, and total glutathione (GSH) content) and fitness-related traits (egg mortality, development dynamics, and mass of nymphs) were monitored in nymphs. The experimental groups were: control (kept out of influence of the magnets), a group exposed to a constant magnetic field (CMF) of 50 mT, and a group exposed to an alternating magnetic field (AMF) of 50 Hz, 6 mT. We found increased SOD and CAT activities in animals exposed to constant and AMFs, whereas GSH activity was not influenced by experimental magnetic fields. No differences were found in egg mortality between control and experimental groups. Significant differences in the time of development between the control and the CMF group were observed, as well as between the CMF and the AMF group. No differences were found in the mass of the nymphs between the three experimental groups. In conclusion, CMF and AMF have the possibility to modulate the antioxidative defense and some of the fitness-related traits in B. extradentatum.

Changes in activity of non-specific esterases in cadmium treated Lymantria dispar larvae.

Many biochemical, physiological and histological criteria have been used as indicators of exposures and effects of the contaminants. These changes can indicate the response of an organism to a specific environmental stressor. In the present paper, the effect of the acute and chronic exposure to cadmium as well as recovery from two cadmium concentrations (10 and 30 µgCd/g dry food) on gypsy moth (Lymantria dispar) midgut esterases was investigated. The influence of cadmium on trait plasticity was also examined. Esterases showed great sensitivity to low metal concentrations during acute and chronic treatments. Their activities during short-term exposure and after recovery significantly depended on cadmium concentrations. The esterases had greater index of plasticity during chronic treatments with 10 and 30 µgCd/dry food. Five esterase isoforms between 64 and 250 kDa were detected. Isoforms of esterases exposed to any of the two cadmium effects differed among several egg-masses. Isozymes were distinguished in one egg-mass during different cadmium treatments. We conclude that these enzymes could be considered potential and sensitive non-selective biomarkers for the presence of cadmium in food.

The effects of temperature stress and population origin on the thermal sensitivity of Lymantria dispar L. (Lepidoptera: Erebidae) larvae.

Increased environmental temperature is one of the most frequent stresses effecting metabolic rate in herbivorous insect species. Our goal was to compare the influence of increased environmental temperature and induced thermotolerance on the activity of midgut phosphatases and brain tissue hsp70 concentration in 5th instar Lymantria dispar larvae originating from an unpolluted and polluted forest. Induced thermotolerance (larval pre-treatment at high, sub-lethal temperature) increases the species ability to overcome the negative effects of thermal stress, therefore we monitored the effect of this regime in larvae originating from both forests. Thermal regimes in this experiment predominantly influenced the alkaline phosphatases activity and it was affected by temperature, population origin, and their combined effect. Total acid phosphatases activity was changed only by the joint effect of temperature and population origin. Brain hsp70 concentration was under a significant individual and joint effect of temperature and population. In both populations, brain tissue hsp70 concentration and alkaline phosphatases activity should be taken under consideration as a battery with biomarker potential for thermal stress in L. dispar larvae as a bioindicator species.

The embryonic and post-embryonic development in two Drosophila species exposed to the static magnetic field of 60 mT.

In this study, a static magnetic field influence on development and viability in two different species, Drosophila melanogaster and Drosophila hydei, was investigated. Both species completed development (egg-adult), in and out of the static magnetic field induced by double horseshoe magnet. Treated vials with eggs were placed in the gap between magnetic poles (47 mm) and exposed to the average magnetic induction of 60 mT, while control ones were kept far enough from magnetic field source. We found that exposure to the static magnetic field reduced development time in both species, but statistical significance was found only for D. hydei. Furthermore, we found that the average viability of both Drosophila species exposed to the magnetic field was significantly weaker compared to control ones. These results indicate that 60 mT static magnetic field could be considered as a potential stressor, influencing on different levels the embryonic and post-embryonic development of individuals.

Implications of long-term exposure of a Lymantria dispar L. population to pollution for the response of larval midgut proteases and acid phosphatases to chronic cadmium treatment.

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.

Sensitivity of midgut physiological parameters of Lymantria dispar L. larvae to benzo[a]pyrene in populations with different multigeneration contact to environmental pollutants.

Accumulation of organic pollutants in the environment calls for sensing physiological parameters adequate to indicate the presence of contaminants and their effects on ecosystems. Evidence points to the importance of insect adaptations in their habitats for the assessment of sensitive biomarkers so we examined the influence of origin and multigenerational adaptations of the Lymantria dispar larvae to chronic benzo[a]pyrene (B[a]P) treatment under laboratory conditions. The main aim was to compare reactions of larvae from unpolluted and polluted forests using alkaline phosphatase (ALP), acid phosphatase (ACP), and carboxylesterase (CE) specific activities in the midgut, including electrophoretic isoform patterns; midgut expression levels of Hsp70, larval development time (DT), and midgut mass (MM), after chronic exposure to 5 and 50 ng of B[a]P/g dry food weight. The biomarker potential of these parameters regarding larval pre-exposure history to pollution was estimated by principal component analysis (PCA). B[a]P treatment resulted in inhibition of ALP activity, a rise of CE activity, and reduction of MM in larvae from the unpolluted forest, while the population from the polluted forest showed significant elevation of Hsp70 expression in the midgut, prolonged DT, and reduction of MM. PCA confirmed variations in responses of the selected parameters regarding population origin. The obtained results provide insight into insect population variability concerning physiological responses to pollutants. It is indicative that all investigated physiological parameters of L. dispar larvae showed origin-dependent responses to long-term presence of B[a]P, which may be of great importance in ecotoxicological research.

Effects of fluoranthene on digestive enzymes activity and relative growth rate of larvae of lepidopteran species, Lymantria dispar L. and Euproctis chrysorrhoea L.

Fluoranthene is one of the most abundant polycyclic aromatic hydrocarbon pollutants in the environment and it may accumulate in plant leaves which are the main food source for phytophagous insect species. The aim of this study was to establish the effects of dietary fluoranthene on specific activities of digestive enzymes and expression of their isoforms in the midgut, and the relative growth rates of Lymantria dispar and Euproctis chrysorrhoea larvae. Exposure to fluoranthene led to significantly decreased trypsin activity in the midgut of larvae of both species. Leucine aminopeptidase activity decreased significantly in the midgut of L. dispar larvae exposed to the lower concentration of fluoranthene, but that enzyme activity showed the opposite trend in E. chrysorrhoea larvae. There was no pollutant induced changes in lipase activity in L. dispar, while elevated enzyme activity was recorded in the midgut of E. chrysorrhoea larvae exposed to the lower concentration of fluoranthene. Different patterns of expression of enzyme isoforms were noticed. Relative growth rates of both species significantly decreased in fluoranthene treated larvae. These responses indicate to the significance of relationships between physiological changes and fitness-related traits in L. dispar and E. chrysorrhoea larvae affected by pollutant, and contribute to understanding the mechanisms of their adjustment to stressful conditions.

The impact of chronic exposure to a magnetic field on energy metabolism and locomotion of Blaptica dubia.

Purpose: This study deals with a comparative analysis of the effects of chronic exposure to a static magnetic field (SMF) and an extremely low frequency magnetic field (ELF MF) in Blaptica dubia nymphs. The outcome of such treatment on insect and fat body mass, glycogen and total lipid content in the fat body and locomotion, as an energy demanding process, were examined.Materials and methods: One-month-old nymphs of B. dubia were exposed to an SMF (110 mT) or ELF MF (50 Hz, 10 mT) for 5 months. Their locomotion was monitored in the 'open-field' test for 10 min and expressed as travel distance, time in movement and average speed while in motion. After that, fat body mass and content of its main components (glycogen and total lipids) were determined. Nymph body mass was also estimated after 1 and 5 months of MF treatment.Results: Chronic exposure to the SMF and ELF MF decreased nymph body mass and glycogen content in the fat body but increased all examined parameters of locomotion. In addition, chronic SMF treatment elevated total lipid content in the fat body, while chronic ELF MF treatment reduced fat body mass and total lipid content.Conclusions: These findings indicate that B. dubia nymphs are sensitive to the applied MFs and possess different strategies for fuel usage in response to the SMF and ELF MF in order to satisfy increased energy demands and to overcome stressful conditions.

Effects of ghrelin on the feeding behavior of Lymantria dispar L. (Lymantriidae) caterpillars.

Ghrelin is a 28-amino acid peptide which has significant effects on animal appetite, thus influencing body mass. The aim of our study was to examine the effects of ghrelin on the feeding behavior and physiology 4th instar caterpillars of the pest insect, Lymantria dispar L. Treatment of 4th instar caterpillars with four subpicomolar amounts of ghrelin had a positive influence on daily food intake, frass elimination, body mass. Also, locomotor activity increased, while stadium duration decreased in treated caterpillars. The similarity between the effects of ghrelin on caterpillar physiology and those in mammals suggests that using this model system for further studies of neuroendocrinological processes underlying feeding could lead to essential information about more complex organisms.