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.

Antioxidative enzymes, alkaline phosphatases and Hsp70 expression in larvae of Lymantria dispar (Lepidoptera: Erebidae) from unpolluted and polluted forests after chronic cadmium treatment.

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

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.

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.

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.

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.

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.

Tissue-specific responses of Lymantria dispar L. (Lepidoptera: Erebidae) larvae from unpolluted and polluted forests to thermal stress.

In this paper the effects of increased environmental temperature on the relative growth rate (RGR) and developmental time in 5th instar L. dispar larvae originating from unpolluted and polluted forests were analyzed. As indicators of the level of generated reactive oxygen species in thermal stress, we estimated midgut and hemolymph activity of the antioxidative enzymes, superoxide dismutase (SOD) and catalase (CAT), as well as the detoxifying enzymes glutathione S-transferase (GST), carboxylesterase (CaE) and acetylcholinesterase (AChE) from the midgut and brain tissue. We also examined the influence of induced thermotolerance as a species' ability to overcome the negative effects of this stressor. In larvae originating from the unpolluted forest, the midgut is the primary location of increased SOD and CAT activity and induced thermotolerance did not modified their activity in either tissue. In larvae from the polluted forest, in both tissues SOD activity was more sensitive to an increased temperature and induced thermotolerance than CAT. Carboxylesterase responded diversely to thermal stress depending on the analyzed tissue regardless the origin of larvae, while the activity of GST and AChE in tissue depended on the origin of larvae. Induced thermotolerance modified the activity of detoxifying enzymes in larvae originating from the polluted forest. Combining the selected parameters into an integrated biomarker response (IBR) the GST, CaE and AChE battery emerged as a potential biomarker for thermal stress in L. dispar larvae.

Parameters of oxidative stress, cholinesterase activity, Cd bioaccumulation in the brain and midgut of Lymantria dispar (Lepidoptera: Lymantriidae) caterpillars from unpolluted and polluted forests.

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

Acetylcholinesterase and heat shock protein 70 response in larval brain tissue of Lymantria dispar L. (Lepidoptera, Limantriidae) upon chronic exposure to benzo(a)pyrene.

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.

Influence of dietary cadmium exposure on fitness traits and its accumulation (with an overview on trace elements) in Lymantria dispar larvae.

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 100µgCd/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.

Effects of benzo[a]pyrene dietary intake to antioxidative enzymes of Lymantria dispar (Lepidoptera: Lymantriidae) larvae from unpolluted and polluted forests.

Anthropogenic activity in industrial development has imposed great threats to the environment and wildlife in the form of persistent organic pollutants. Polycyclic aromatic hydrocarbons (PAH) tend to accumulate in vegetation foliage which is the main food source of polyphagous insect species Lymantria dispar L. Origin and multigenerational adaptation of L. dispar population to environmental challenges strongly condition the enzymes' sensitivity to pollutants. In this study, our aim was to investigate response of the superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) to the chronic dietary exposure of benzo[a]pyrene in the midgut tissues and hemolymph of two L. dispar populations originating from unpolluted and polluted forest habitat. Midgut tissue of the larvae from the polluted forest showed significant increase in SOD, CAT and GST activity, while in unpolluted forest's larvae SOD and CAT showed elevated activities in hemolymph. L. dispar populations adapted to different level of pollution in their environment and expressed distinct tissue-dependent antioxidative enzyme sensitivity to benzo[a]pyrene diet, implying high potential for further elucidation of these enzymes as molecular biomarkers.

Genotoxic effects of cadmium and influence on fitness components of Lymantria dispar caterpillars.

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.

Acetylcholinesterase (AChE) and heat shock proteins (Hsp70) of gypsy moth (Lymantria dispar L.) larvae in response to long-term fluoranthene exposure.

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.

Glutathione S-transferase in the midgut tissue of gypsy moth (Lymantria dispar) caterpillars exposed to dietary cadmium.

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.

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.

Cadmium-induced changes of gypsy moth larval mass and protease activity.

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

Enzymatic biomarkers as indicators of dietary cadmium in gypsy moth caterpillars.

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.

Improved regeneration after spinal cord injury in mice lacking functional T- and B-lymphocytes.

It is widely accepted that the immune system plays important functional roles in regeneration after injury to the spinal cord. Immune response towards injury involves a complex interplay of immune system cells, such as neutrophils, macrophages and microglia, T- and B-lymphocytes. We investigated the influence of the lymphocyte component of the immune system on the locomotor outcome of severe spinal cord injury in a genetic mouse model of immune suppression. Transgenic mice lacking mature T- and B-lymphocytes due to the recombination activating gene 2 gene deletion (RAG2-/- mice) were subjected to severe compression of the lower thoracic spinal cord, with the wild-type mice of the same inbred background serving as controls. According to both the Basso Mouse Scale score and single frame motion analysis, the RAG2-/- mice showed improved recovery in comparison to control mice at six weeks after injury. Better locomotor function was associated with enhanced catecholaminergic and cholinergic reinnervation of the spinal cord caudal to injury and increased axonal regrowth/sprouting at the site of injury. Myelination of axons in the ventral column measured as g-ratio was more extensive in RAG2-/- than in control mice 6weeks after injury. Additionally, the number of microglia/macrophages was decreased in the lumbar spinal cord of RAG2-/- mice after injury, whereas the number of astrocytes was increased compared with controls. We conclude that T- and B-lymphocytes restrict functional recovery from spinal cord injury by increasing numbers of microglia/macrophages as well as decreasing axonal sprouting and myelination.