Repellent activity of Tanacetum parthenium (L.) and Tanacetum vulgare (L.) essential oils against Leptinotarsa decemlineata (Say).

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is one of the most destructive pest species to have developed resistance to most chemical insecticides. We determined the composition and evaluated the potential of Tanacetum parthenium L. and Tanacetum vulgare L. (Asteraceae family) essential oil (EO) application as an alternative eco-friendly control strategy against L. decemlineata. We assessed the antifeedant activity for L. decemlineata larvae and adults by estimating the damage to potato leaves treated with three concentrations of EOs dissolved in ethanol (0.125, 0.25 and 0.5%). Results showed that T. parthenium EO was more effective against larvae, and T. vulgare was more effective against adults. In an olfactometer assay, the time required to choose an untreated leaf disc did not depend on the Tanacetum species, or life stage examined. However, the concentration of EO exhibited a significant effect on the behaviour of both developmental stages. At higher EO concentrations, both third instar larvae and adults require less time to choose an untreated leaf disc. Additionally, T. parthenium EO provoked more rapid movement away from the treated leaf disc than T. vulgare, especially at the highest concentration. Successful modification of L. decemlineata behaviour by the two Tanacetum oils suggests that they possess the potential for use in potato protection.

Experimentally induced host-shift changes life-history strategy in a seed beetle.

Expansion of the host range in phytophagous insects depends on their ability to form an association with a novel plant through changes in host-related traits. Phenotypic plasticity has important effects on initial survival of individuals faced with a new plant, as well as on the courses of evolutionary change during long-term adaptation to novel conditions. Using experimental populations of the seed beetle that evolved on ancestral (common bean) or novel (chickpea) host and applying reciprocal transplant at both larval and adult stage on the alternative host plant, we studied the relationship between the initial (plastic) phases of host-shift and the subsequent stages of evolutionary divergence in life-history strategies between populations exposed to the host-shift process. After 48 generations, populations became well adapted to chickpea by evolving the life-history strategy with prolonged larval development, increased body mass, earlier reproduction, shorter lifespan and decreased plasticity of all traits compared with ancestral conditions. In chickpea-adapted beetles, negative fitness consequences of low plasticity of pre-adult development (revealed as severe decrease in egg-to-adult viability on beans) exhibited mismatch with positive effects of low plasticity (i.e. low host sensitivity) in oviposition and fecundity. In contrast, beetles adapted to the ancestral host showed high plasticity of developmental process, which enabled high larval survival on chickpea, whereas elevated plasticity in adult behaviour (i.e. high host sensitivity) resulted in delayed reproduction and decreased fecundity on chickpea. The analysis of population growth parameters revealed significant fluctuation during successive phases of the host-shift process in A. obtectus.

Extraordinary Adaptive Plasticity of Colorado Potato Beetle: "Ten-Striped Spearman" in the Era of Biotechnological Warfare.

Expanding from remote areas of Mexico to a worldwide scale, the ten-striped insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata Say), has risen from being an innocuous beetle to a prominent global pest. A diverse life cycle, phenotypic plasticity, adaptation to adverse conditions, and capability to detoxify or tolerate toxins make this insect appear to be virtually "indestructible". With increasing advances in molecular biology, tools of biotechnological warfare were deployed to combat CPB. In the last three decades, genetically modified potato has created a new challenge for the beetle. After reviewing hundreds of scientific papers dealing with CPB control, it became clear that even biotechnological means of control, if used alone, would not defeat the Colorado potato beetle. This control measure once again appears to be provoking the potato beetle to exhibit its remarkable adaptability. Nonetheless, the potential for adaptation to these techniques has increased our knowledge of this pest and thus opened possibilities for devising more sustainable CPB management programs.

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.

The effect of mitochondrial complex I inhibitor on longevity of short-lived and long-lived seed beetles and its mitonuclear hybrids.

Mitochondria are suggested to play a central role in ageing and evolution of longevity. Gradual decline in mitochondrial function during ageing and concomitant increase in production of reactive oxygen species (ROS) leads to oxidative damage of macromolecules and impairment of ATP synthesis. To assess relationship between ageing and oxidative stress resistance we exposed different longevity lines of the seed beetle (Acanthoscelides obtectus) to four concentrations of tebufenpyrad, mitochondrial complex I inhibitor. Complex I is one of main sites of ROS production during normal respiration and its inhibition elevates oxidative stress. Our results showed that 24 h of exposure to tebufenpyrad decreased survival and post-stress longevity due to increased baseline mortality. Higher resistance was recorded in beetles from lines selected for late reproduction and extended longevity (L) than in early reproducing beetles (E). Also, females were more resistant than males. Since complex I is under dual genetic control, our second aim was to disentangle relative contribution of nuclear and mitochondrial genes to the variation in longevity. We used crossed combinations of distinct mitochondrial and nuclear genotypes (E × L, L × E) and compared them to control hybrids where mitochondrial genome was "transplanted" onto the original background (E × E, L × L). Our study revealed significant effect of nucleus, i.e. higher survival and post-stress longevity in beetles harbouring L nucleus. Mitochondrion effect was significant only within L nuclear background where E mitochondrion gave advantage.

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.

Resistance to prooxidant agent paraquat in the short- and long-lived lines of the seed beetle (Acanthoscelides obtectus).

In the present study we test whether variation in resistance to paraquat (PQ), a free radical generator, correlates with variation in longevity in two sets of seed beetles (Acanthoscelides obtectus) experimental lines that were selected either for early reproduction and short-life or late reproduction and long-life. Long-lived late reproduction lines (L) showed increased resistance to PQ, while opposite was true for short-lived early reproduction line (E). Striking outcome of the selection for early and late reproduction in A. obtectus is asymmetry of responses to alternate mating schedules. The intensity of response depended on selection regime, sex and PQ dose. Evolution of longevity and PQ resistance was faster in L than E selection regime, and in females than males. To understand how age-specific mortality rates are affected by PQ we decomposed post-stress mortality data (using Gompertz mortality model) into initial mortality rate, which reflects basal vulnerability to stresses and age-specific mortality rate, which concerns the rate of increase in stress vulnerability, i.e. the rate of senescence. By estimating the parameters of the Gompertz mortality model we have shown that longevity reduction caused by PQ was the consequence of the increased baseline mortality rather than a speed up of the rate of ageing.

Temperature- and Diet-Induced Plasticity of Growth and Digestive Enzymes Activity in Spongy Moth Larvae.

Temperature and food quality are the most important environmental factors determining the performance of herbivorous insects. The objective of our study was to evaluate the responses of the spongy moth (formerly known as the gypsy moth) [Lymantria dispar L. (Lepidoptera: Erebidae)] to simultaneous variation in these two factors. From hatching to the fourth instar, larvae were exposed to three temperatures (19 °C, 23 °C, and 28 °C) and fed four artificial diets that differed in protein (P) and carbohydrate (C) content. Within each temperature regime, the effects of the nutrient content (P+C) and ratio (P:C) on development duration, larval mass, growth rate, and activities of digestive proteases, carbohydrases, and lipase were examined. It was found that temperature and food quality had a significant effect on the fitness-related traits and digestive physiology of the larvae. The greatest mass and highest growth rate were obtained at 28 °C on a high-protein low-carbohydrate diet. A homeostatic increase in activity was observed for total protease, trypsin, and amylase in response to low substrate levels in the diet. A significant modulation of overall enzyme activities in response to 28 °C was detected only with a low diet quality. A decrease in the nutrient content and P:C ratio only affected the coordination of enzyme activities at 28 °C, as indicated by the significantly altered correlation matrices. Multiple linear regression analysis showed that variation in fitness traits in response to different rearing conditions could be explained by variation in digestion. Our results contribute to the understanding of the role of digestive enzymes in post-ingestive nutrient balancing.

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.

Pure Camphor and a Thujone-Camphor Mixture as Eco-Friendly Antifeedants against Larvae and Adults of the Colorado Potato Beetle.

The Colorado potato beetle (CPB) is a serious pest of economically important Solanaceae species. The use of essential oil compounds in pest management has been proposed as an alternative to harmful chemical insecticides that disturb human health and ecosystem functioning. We examined the antifeedant activity of three concentrations (0.125%, 0.25% and 0.5%) of pure camphor and a thujone-camphor mixture against 3rd instar larvae and adults. Their efficacy was evaluated according to the degree of leaf damage and avoidance of treated leaves by the CPB. Treatment of potato leaves significantly reduced leaf damage compared to the control. Leaf protection increased at higher concentrations of the examined compounds. Camphor was more effective against larvae and the thujone-camphor mixture was more effective against adults. Additionally, adults moved faster towards the control leaf disc in the two-choice olfactometer assay if an alternative disc was treated with a thujone-camphor mixture, whereas larvae responded similarly to the two potential repellents. However, after contact with the leaf disc treated with the highest compound concentration, the larvae escaped faster from the thujone-camphor mixture than from pure camphor. In conclusion, both examined compounds are promising eco-friendly antifeedants, but their efficacy depends on the developmental stage of the beetle, compound type and applied concentration.

Assessment of Sex-Specific Toxicity and Physiological Responses to Thymol in a Common Bean Pest Acanthoscelides obtectus Say.

Acanthoscelides obtectus Say (Coleoptera: Chrysomelidae: Bruchinae), is one of the most important pests of the common bean Phaseolus vulgaris L. Without appropriate management it may cause significant seed loss in storages. In search for means of environmentally safe and effective protection of beans we assessed biological activity of thymol, an oxygenated monoterpene present in essential oils of many aromatic plants. We studied contact toxicity of thymol on bean seeds and its effects on adult longevity and emergence in F1 generation. Furthermore, we determined acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), mixed-function oxidase (MFO), carboxylesterases (CarE) and glutathione S-transferase (GST) activities in response to 24 h exposure of beetles to sublethal and lethal thymol concentrations. Our results showed that thymol decreased adult survival, longevity and percentage of adult emergence. Higher median lethal concentration (LC50) was recorded in females indicating their higher tolerance comparing to males. Overall, activities of SOD, CAT and CarE increased at sublethal and MFO increased at both sublethal and lethal thymol concentrations. On the other hand, GST and AChE activities decreased along with the increase in thymol concentrations from sublethal (1/5 of LC50, 1/2 of LC50) to lethal (LC50). Enzyme responses to the presence of thymol on bean seed were sex-specific. In the control group females had lower CarE and higher SOD, CAT and GST activity than males. In treatment groups, females had much higher CAT activity and much lower CarE activity than males. Our results contribute to deeper understanding of physiological mechanisms underlying thymol toxicity and tolerance which should be taken into account in future formulation of a thymol-based insecticide.

Potential of Essential Oils from Anise, Dill and Fennel Seeds for the Gypsy Moth Control.

The gypsy moth (Lymantria dispar L. (Lepidoptera: Erebidae)) is a serious pest of hardwood forests. In the search for an environmentally safe means of its control, we assessed the impact of different concentrations of essential oils (EOs) from the seeds of three Apiaceae plants (anise Pimpinella anisum, dill Anethum graveolens, and fennel Foeniculum vulgare) on behavior, mortality, molting and nutritional physiology of gypsy moth larvae (GML). EOs efficacy was compared with commercial insecticide NeemAzal®-T/S (neem). The main compounds in the Eos were trans-anethole in anise; carvone, limonene, and α-phellandrene in dill; and trans-anethole and fenchone in fennel seed. At 1% EOs concentration, anise and fennel were better antifeedants and all three EOs were more toxic than neem. Neem was superior in delaying 2nd to 3rd larval molting. In the 4th instar, 0.5%, anise and fennel EOs decreased relative consumption rate more than neem, whereas all three EOs were more effective in reducing growth rate, approximate digestibility and efficiency of conversion of food into body mass leading to higher metabolic costs to GML. Decrease in consumption and metabolic parameters compared to control GML confirmed that adverse effects of the EOs stem from both pre- and post-ingestive mechanisms. The results indicate the potential of three EOs to be used for gypsy moth control.

Effects of cadmium on protocerebral neurosecretory neurons and fitness components in Lymantria dispar L.

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

Evaluation of tansy essential oil as a potential "green" alternative for gypsy moth control.

The development of "green" alternatives to chemical pesticides could play a crucial role in integrated pest management (IPM). Their use is considered either as a substitution for or in addition to hazardous synthetic products. We analysed the influence of three concentrations of tansy (Tanacetum vulgare L.) essential oil (EO), previously characterised by GC-MS, on the survival and moulting of the 2nd instar and the nutritional indices of the 4th instar gypsy moth (Lymantria dispar L.) larvae. In a residual contact toxicity assessment, the exposure to tansy EO caused low mortality (< 10%) while larval development was significantly slowed down, i.e., the percentage of larvae that moulted into the 3rd instar was reduced. On the other hand, when tansy EO was incorporated into the diet (digestive toxicity assay), high mortality and a lack of moulting after 120 h of eating were recorded for the highest applied concentration of EO. During 48 h of feeding on EO-supplemented food at concentrations of 0.5 and 1% (v/v), the relative growth rate (RGR) of the 4th instar larvae significantly decreased, which can be explained by a significant reduction of the relative consumption rate (RCR) and significantly or marginally significantly lower efficiency of conversion of ingested food into insect biomass (ECI). Although the RCR was also reduced with the lowest applied EO concentration (0.1%), the ECI was not affected which meant the RGR was as high as it was for the control larvae. ECI changes, when two higher EO concentrations were applied, were due to a reduction in the efficiency of conversion of digested food into biomass (ECD), while approximate digestibility was unaffected by the presence of EO in the food. Our results on the significant negative effects of tansy EO on gypsy moth larval survival, development time, and nutritional physiology suggest that it could be considered in future designs for botanical insecticides for gypsy moth control.

Toxic, Oviposition Deterrent and Oxidative Stress Effects of Thymus vulgaris Essential Oil against Acanthoscelides obtectus.

The bean weevil Acanthoscelides obtectus Say (Coleoptera: Chrysomelidae: Bruchinae) can cause significant losses in production of its primary host common bean Phaseolus vulgaris L. To avoid bean protection with environmentally risky chemical insecticides and provide sustainable and safe production of food, new pest management methods based on natural compounds are investigated. In the present study, we evaluated protective potential of the essential oil (EO) from the common thyme Thymus vulgaris L. applied on bean seeds. We assessed residual contact toxicity of thyme EO and its effects on A. obtectus longevity, oviposition and adult emergence. Furthermore, to elucidate the role of oxidative stress in thyme EO toxicity, we estimated the levels of oxidatively damaged proteins and lipids, as well as the level of thiols which have important role for antioxidant capacity. We found that thyme oil significantly reduced adult survival and longevity, induced oxidative damage to lipids and proteins and depleted protein and non-protein thiols in a concentration-dependent manner. Females appeared to be more tolerant to thyme oil treatment than males. Sublethal EO concentrations affected oxidative stress indices, deterred oviposition and strongly inhibited adult emergence. The results suggest that thyme oil has the potential to be used as an ecofriendly insecticide for A. obtectus control.

Plastic responses of larval mass and alkaline phosphatase to cadmium in the gypsy moth larvae.

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

Partial characterization of a lipase from gypsy moth (lymantria dispar L.) larval midgut.

Lipase activity of the gypsy moth (Lymantria dispar L.) was studied by the spectrophotometric method using crude homogenate of fifth-instar larval midgut tissues as the enzyme source and p-nitrophenyl caprylate (pNPC) as substrate. A Km value of 0.310 mM and a Vmax value of 1.479 U/mg prot. were obtained for this substrate. Among various p-nitrophenyl esters tested, maximum activity was obtained for p-nitrophenyl caprylate and p-nitrophenyl caprate. The enzyme was most active at alkaline pH, with maximum at pH 8.2. Decreased activity was detected after preincubation in buffers of pH below 7.0 and above 8.2. The enzyme was unstable at room temperature. The enzyme was Ca2+ independent. Its activity was inhibited by PMSF, Fe2+, Ag+ and Pb2+, while Fe3+ inhibited enzyme activity by about 40%.

Co-expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development.

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Dragacevka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long-term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.

Sex-specific mitonuclear epistasis and the evolution of mitochondrial bioenergetics, ageing, and life history in seed beetles.

The role of mitochondrial DNA for the evolution of life-history traits remains debated. We examined mitonuclear effects on the activity of the multisubunit complex of the electron transport chain (ETC) involved in oxidative phosphorylation (OXPHOS) across lines of the seed beetle Acanthoscelides obtectus selected for a short (E) or a long (L) life for more than >160 generations. We constructed and phenotyped mitonuclear introgression lines, which allowed us to assess the independent effects of the evolutionary history of the nuclear and the mitochondrial genome. The nuclear genome was responsible for the largest share of divergence seen in ageing. However, the mitochondrial genome also had sizeable effects, which were sex-specific and expressed primarily as epistatic interactions with the nuclear genome. The effects of mitonuclear disruption were largely consistent with mitonuclear coadaptation. Variation in ETC activity explained a large proportion of variance in ageing and life-history traits and this multivariate relationship differed somewhat between the sexes. In conclusion, mitonuclear epistasis has played an important role in the laboratory evolution of ETC complex activity, ageing, and life histories and these are closely associated. The mitonuclear architecture of evolved differences in life-history traits and mitochondrial bioenergetics was sex-specific.

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.