Cameraria Ohridella Research Articles

The role of geography and host abundance in the distribution of parasitoids of an alien pest

Chalcid wasps (Hymenoptera: Chalcidoidea) are probably the most effective and abundant parasitoids of the horse chestnut leaf miner (Cameraria ohridella), an alien pest in Europe that lacks specialized enemies. We studied how the species richness and abundance of chalcids are influenced by altitude, direction of an alien spread and host abundance of C. ohridella. We quantified the numbers and species richness of chalcid wasps and the numbers of C. ohridella that emerged from horse chestnut (Aesculus hippocastanum) leaf litter samples collected from 35 sites in the Czech Republic. Species richness of chalcids, which was considered an indicator of the possible adaptation of parasitoids to this alien host, was unrelated to C. ohridella abundance, direction of spread, or altitude. Chalcid abundance, which was considered an indicator of parasitism of the alien host, was strongly and positively related to C. ohridella abundance. Chalcid abundance was negatively related to direction of spread and positively related, although in a non-linear manner, to altitude. The relationship of chalcid abundance with direction of spread and altitude was weaker than that with C. ohridella abundance. The results provide evidence that biological control of the alien pest C. ohridella by natural enemies might develop in the future.

Assessing the potential distribution of insect pests: case studies on large pine weevil (Hylobius abietis L) and horse‐chestnut leaf miner (Cameraria ohridella) under present and future climate conditions in European forests

Forest insect pests represent a serious threat to European forests and their negative effects could be exacerbated by climate change. This paper illustrates how species distribution modelling integrated with host tree species distribution data can be used to assess forest vulnerability to this threat. Two case studies are used: large pine weevil (Hylobius abietis L) and horse‐chestnut leaf miner (Cameraria ohridella Deschka & Dimič) both at pan‐European level. The proposed approach integrates information from different sources. Occurrence data of insect pests were collected from the Global Biodiversity Information Facility (GBIF), climatic variables for present climate and future scenarios were sourced, respectively, from WorldClim and from the Research Program on Climate Change, Agriculture and Food Security (CCAFS), and distributional data of host tree species were obtained from the European Forest Data Centre (EFDAC), within the Forest Information System for Europe (FISE). The potential habitat of the target pests was calculated using the machine learning algorithm of Maxent model. On the one hand, the results highlight the potential of species distribution modelling as a valuable tool for decision makers. On the other hand, they stress how this approach can be limited by poor pest data availability, emphasizing the need to establish a harmonised open European database of geo‐referenced insect pest distribution data.

Evaluating methods to quantify spatial variation in the velocity of biological invasions

Invading species rarely spread homogeneously through a landscape and invasion patterns typically display irregular frontal boundaries as the invasion progresses through space. Those irregular patterns are generally produced by local environmental factors that may slow or accelerate movement of the frontal boundary. While there is an abundant literature on species distribution modelling methods that quantify local suitability for species establishment, comparatively few studies have examined methods for measuring the local velocity of invasions that can then be statistically analysed in relation to spatially variable environmental factors. Previous studies have used simulations to compare different methods for estimating the overall rate of spread of an invasion. We adopted a similar approach of simulating invasions that resemble two real case‐studies, both in terms of their spatial resolution (i.e. considering the size of one cell as one km) and their spatial extent (> 600 000 km²). Simulations were sampled to compare how different methods used to measure local spread rate, namely the neighbouring, nearest distance and Delaunay methods, perform for spatio‐temporal comparisons. We varied the assessment using three levels of complexity of the spatio‐temporal pattern of invasion, three sample sizes (500, 1000 and 2000 points), three different spatial sampling patterns (stratified, random, aggregated), three interpolation methods (generalized linear model, kriging, thin plate spline regression) and two spatio‐temporal modelling structures (trend surface analysis and boundary displacement), resulting in a total of 486 different scenarios. The thin plate spline regression interpolation method, in combination with trend surface analysis, was found to provide the most robust local spread rate quantification as it was able to reliably accommodate different sampling conditions and invasion patterns. This best approach was successfully applied to two case‐studies, the invasion of France by the horse‐chestnut leafminerCameraria ohridellaand by the bluetongue virus, generally in agreement with previously published values of spread rates. Potential avenues for further research are discussed.

Biological object recognition in μ-radiography images

This study presents an applicability of real-time microradiography to biological objects, namely to horse chestnut leafminer, Cameraria ohridella (Insecta: Lepidoptera, Gracillariidae) and following image processing focusing on image segmentation and object recognition. The microradiography of insects (such as horse chestnut leafminer) provides a non-invasive imaging that leaves the organisms alive. The imaging requires a high spatial resolution (micrometer scale) radiographic system. Our radiographic system consists of a micro-focus X-ray tube and two types of detectors. The first is a charge integrating detector (Hamamatsu flat panel), the second is a pixel semiconductor detector (Medipix2 detector). The latter allows detection of single quantum photon of ionizing radiation. We obtained numerous horse chestnuts leafminer pupae in several microradiography images easy recognizable in automatic mode using the image processing methods. We implemented an algorithm that is able to count a number of dead and alive pupae in images. The algorithm was based on two methods: 1) noise reduction using mathematical morphology filters, 2) Canny edge detection. The accuracy of the algorithm is higher for the Medipix2 (average recall for detection of alive pupae =0.99, average recall for detection of dead pupae =0.83), than for the flat panel (average recall for detection of alive pupae =0.99, average recall for detection of dead pupae =0.77). Therefore, we conclude that Medipix2 has lower noise and better displays contours (edges) of biological objects. Our method allows automatic selection and calculation of dead and alive chestnut leafminer pupae. It leads to faster monitoring of the population of one of the world's important insect pest.

Ultrastructural and biochemical comparison of summer active and summer diapausing pupae of the horse chestnut leaf miner, Cameraria ohridella (Lepidoptera: Gracillariidae)

The ultrastructural and biochemical characteristics were studied in active and diapausing pupae of the horse chestnut leaf miner, Cameraria ohridella, collected in summer. The active pupae contained developing internal organs, while those in diapause mostly numerous non-differentiated cells packed with stored nutrients. These nutrients occurred in vacuoles as liquid material (lipids, proteins) or as reserve crystaloids (proteins). Triacylglycerols (TGs) were the main lipids stored by pupae and were more abundant in diapausing than active pupae. The amount of diacyglycerols (DGs) and phospholipids (PLs) was almost identical in both groups, which reflects the roles of these compounds in lipid transport and structure of cell membranes, respectively. A principal component analysis (PCA) indicated differences in the quality of the lipid compounds in both groups and that the tgs were mostly responsible for the dif- ference. Polyunsaturated linolenic acid (18 : 3) was the most abundant fatty acid in both active and diapausing pupae, nevertheless its content was significantly higher in the former; the level of 7 fatty acids reached values higher than 1%. Differences in other nutrients were similar to those of TGs; higher contents of both glycogen and proteins were recorded in diapausing than active pupae. The results revealed significant differences between the two summer pupal forms of C. ohridella and furthered our understanding of the develop- mental strategy of this successful, invasive pest insect.

A Burst of miRNA Innovation in the Early Evolution of Butterflies and Moths.

MicroRNAs (miRNAs) are involved in posttranscriptional regulation of gene expression. Because several miRNAs are known to affect the stability or translation of developmental regulatory genes, the origin of novel miRNAs may have contributed to the evolution of developmental processes and morphology. Lepidoptera (butterflies and moths) is a species-rich clade with a well-established phylogeny and abundant genomic resources, thereby representing an ideal system in which to study miRNA evolution. We sequenced small RNA libraries from developmental stages of two divergent lepidopterans, Cameraria ohridella (Horse chestnut Leafminer) and Pararge aegeria (Speckled Wood butterfly), discovering 90 and 81 conserved miRNAs, respectively, and many species-specific miRNA sequences. Mapping miRNAs onto the lepidopteran phylogeny reveals rapid miRNA turnover and an episode of miRNA fixation early in lepidopteran evolution, implying that miRNA acquisition accompanied the early radiation of the Lepidoptera. One lepidopteran-specific miRNA gene, miR-2768, is located within an intron of the homeobox gene invected, involved in insect segmental and wing patterning. We identified cubitus interruptus (ci) as a likely direct target of miR-2768, and validated this suppression using a luciferase assay system. We propose a model by which miR-2768 modulates expression of ci in the segmentation pathway and in patterning of lepidopteran wing primordia.

Ancient expansion of the hox cluster in lepidoptera generated four homeobox genes implicated in extra-embryonic tissue formation.

Gene duplications within the conserved Hox cluster are rare in animal evolution, but in Lepidoptera an array of divergent Hox-related genes (Shx genes) has been reported between pb and zen. Here, we use genome sequencing of five lepidopteran species (Polygonia c-album, Pararge aegeria, Callimorpha dominula, Cameraria ohridella, Hepialus sylvina) plus a caddisfly outgroup (Glyphotaelius pellucidus) to trace the evolution of the lepidopteran Shx genes. We demonstrate that Shx genes originated by tandem duplication of zen early in the evolution of large clade Ditrysia; Shx are not found in a caddisfly and a member of the basally diverging Hepialidae (swift moths). Four distinct Shx genes were generated early in ditrysian evolution, and were stably retained in all descendent Lepidoptera except the silkmoth which has additional duplications. Despite extensive sequence divergence, molecular modelling indicates that all four Shx genes have the potential to encode stable homeodomains. The four Shx genes have distinct spatiotemporal expression patterns in early development of the Speckled Wood butterfly (Pararge aegeria), with ShxC demarcating the future sites of extraembryonic tissue formation via strikingly localised maternal RNA in the oocyte. All four genes are also expressed in presumptive serosal cells, prior to the onset of zen expression. Lepidopteran Shx genes represent an unusual example of Hox cluster expansion and integration of novel genes into ancient developmental regulatory networks.

The content of phenolic compounds in leaf tissues of white (Aesculus hippocastanum L.) and red horse chestnut (Aesculus carea H.) colonized by the horse chestnut leaf miner (Cameraria ohridella Deschka & Dimić).

Normally, plant phenolics are secondary metabolites involved in the defense mechanisms of plants against fungal pathogens. Therefore, in this study we attempted to quantify and characterize phenolic compounds in leaves of white and red horse chestnut with leaf miner larvae before and after Cameraria ohridella attack. A total of 17 phenolic compounds belonging to the hydroxycinnamic acid, flavan-3-ols and flavonol groups were identified and quantified in white and red horse chestnut leaf extracts. Significantly decreased concentrations of some phenolic compounds, especially of flavan-3-ols, were observed in infected leaves compared to the non-infected ones. Additionally, a higher content of polyphenolic compounds especially (−)-epicatechin and procyanidins in leaves of red-flowering than in white-flowering horse chestnut may explain their greater resistance to C. ohridella insects.

Leaf traits influencing oviposition preference and larval performance of Cameraria ohridella on native and novel host plants

AbstractLeaf traits are known to influence the preference and performance of phytophagous insects. Traits that influence oviposition can differ from traits that favour larval development, but in native hosts the association between traits usually leads to positive preference‐performance relationships. However, when herbivores interact with novel hosts, traits that influence oviposition and successful larval development can become decoupled, leading to poor preference‐performance relationships. We investigated traits associated with preference and performance of the horse‐chestnut leaf miner, Cameraria ohridella Deschka & Dimic (Lepidoptera: Gracillariidae), on its native host, Aesculus hippocastanum L. (Sapindaceae), and on other species of Aesculus that are novel hosts for the leaf miner. Cameraria ohridella laid eggs on all species of Aesculus, but showed preferences for A. hippocastanum and the phylogenetically closely related Japanese horse‐chestnut, Aesculus turbinata Blume. Successful mine development, however, was observed only on A. hippocastanum and A. turbinata, which indicates a partial mismatch between female oviposition and larval performance. The two susceptible Aesculus species were very similar in their leaf traits, which might explain why larvae performed equally well on these species. In contrast, species resistant to C. ohridella [Aesculus chinensis Bunge, Aesculus indica (Camb) Hook, and Aesculus flava Aiton] showed considerable variation in leaf traits, which suggests that different species of Aesculus might have different mechanisms of resistance to C. ohridella, supporting the idea of different defensive syndromes in different species of plants.

The Success of the Horse-Chestnut Leaf-Miner, Cameraria ohridella, in the UK Revealed with Hypothesis-Led Citizen Science

Citizen science is an increasingly popular way of undertaking research and simultaneously engaging people with science. However, most emphasis of citizen science in environmental science is on long-term monitoring. Here, we demonstrate the opportunities provided by short-term hypothesis-led citizen science. In 2010, we ran the ‘Conker Tree Science’ project, in which over 3500 people in Great Britain provided data at a national scale of an insect (horse-chestnut leaf-mining moth, Cameraria ohridella) undergoing rapid range-expansion. We addressed two hypotheses, and found that (1) the levels of damage caused to leaves of the horse-chestnut tree, Aesculus hippocastanum, and (2) the level of attack by parasitoids of C. ohridella larvae were both greatest where C. ohridella had been present the longest. Specifically there was a rapid rise in leaf damage during the first three years that C. ohridella was present and only a slight rise thereafter, while estimated rates of parasitism (an index of true rates of parasitism) increased from 1.6 to 5.9% when the time C. ohridella had been present in a location increased from 3 to 6 years. We suggest that this increase is due to recruitment of native generalist parasitoids, rather than the adaptation or host-tracking of more specialized parasitoids, as appears to have occurred elsewhere in Europe. Most data collected by participants were accurate, but the counts of parasitoids from participants showed lower concordance with the counts from experts. We statistically modeled this bias and propagated this through our analyses. Bias-corrected estimates of parasitism were lower than those from the raw data, but the trends were similar in magnitude and significance. With appropriate checks for data quality, and statistically correcting for biases where necessary, hypothesis-led citizen science is a potentially powerful tool for carrying out scientific research across large spatial scales while simultaneously engaging many people with science.

Studies of the interaction between horse chestnut leaf miner (Cameraria ohridella) and bacterial bleeding canker (Pseudomonas syringae pv. aesculi)

The white flowering horse chestnut tree (Aesculus hippocastanum L.) was first introduced to the UK ca. 500 years ago. Over the past eight years however, this tree has suffered from severe attack by a mining insect pest known as the horse chestnut leaf miner (HCLM; Cameraria ohridella) and, concomitantly a gram negative bacterium (Pseudomonas syringae pv. aesculi; Pae). Although studies have investigated the influence of each problem individually on tree growth and vitality the interaction between HCLM and Pae on horse chestnut remains unknown. For this reason four year old horse chestnut seedlings were artificially inoculated with Pae in the presence and absence of HCLM. Effects on tree vitality were assessed by monitoring alterations to leaf chlorophyll content, leaf chlorophyll fluorescence (Fv/Fm ratios) as well as key defensive enzymatic activity (β-1,3-glucanase, peroxidase) of woody tissue at the site of Pae infection. With respect to mean lesion size, the main proxy of Pae success or aggressiveness, lesion size was significantly increased in the presence of HCLM compared to trees inoculated with Pae but where HCLM was controlled using insecticide sprays. Results of this study also indicate that suppression of two key defensive enzymes, β-1,3-glucanase and peroxidase, within woody tissue at the site of Pae infection, possibly as a result of reduced photosynthetic productivity caused by HCLM defoliation, maybe the underlying reason as to why Pae severity is greater in the presence of HCLM.

Direct and Unbiased Information Recovery from Liquid Chromatography–Mass Spectrometry Raw Data for Phenotype-Differentiating Metabolites Based on Screening Window Coefficient of Ion Currents

A reworking of a data mining strategy, in which statistical treatment of raw data from liquid chromatography-mass spectrometry (LC-MS) precedes recognition of chromatographic peaks, is presented. In this algorithm the tR-m/z plane of LC-MS data is divided into equal-sized segments of twelve seconds by one m/z unit each, and the total ion currents in corresponding segments as specified by the tR-m/z pair from multiple LC-MS runs are evaluated to generate mean ion currents (μ) and standard deviations (σ). The μ's and σ's of the segments, derived from contrasting classes of LC-MS data set (e.g., resistant-susceptible, case-control, etc.), are used to calculate the Z-factor (screening window coefficient) which is in turn used to rank the segments. Chromatographic peaks are recognized only where the ion currents are shown to differentiate the classes. The result-reporting format enables detection of positive as well as negative correlations between ion intensities and biological traits under study and thus points to the presence of potentially phenotype-discriminating metabolites. Examples of data analyses are presented, in which ions that may distinguish resistant and susceptible species of Aesculus to the leaf-miner Cameraria ohridella were detected.

Parasitoids and parasitism rates of the horse chestnut leaf miner Cameraria ohridella Deschka and Dimić [Lepidoptera: Gracillariidae] across four sites in south-west London

The horse chestnut leaf miner Cameraria ohridella was discovered in the Balkans in 1986 and appeared in the UK in 2002. The aim of this study was to quantify and describe the parasitism rates and parasitoids of the moth in urban and rural stands of Aesculus hippocastanum in the UK and to compare the results with those from European studies. A maximum parasitism rate of 6.67% was obtained, and a significant difference in parasitism rate between urban and rural stands was noted. Two species Minotetrastichus frontalis and Pnigalio agraules alone were responsible for 37% of total parasitism. However, the results show no ideal candidate for use in the biological control of C. ohridella within the native UK parasitoids.

Genetic variation of horse chestnut and red horse chestnut and trees susceptibility to Erysiphe flexuosa and Cameraria ohridella

Trees of Aesculus sp. are often present in European landscapes. Common horse chestnut and red horse chestnut are seriously damaged by the fungus Erysiphe flexuosa as well as the pest Cameraria ohridella. Both pathogen and pest have spread throughout Europe. A genetic background of trees resistance still remains unknown. The present study was undertaken to estimate genetic variation of A. hippocastanum and A.×carnea grown in urban greens and their susceptibility to powdery mildew and horse chestnut leafminer. According to obtained results both species were infected by E. flexuosa in a similar degree but there were significant differences in susceptibility of particular trees (ANOVA). C. ohridella damaged only A. hippocastanum. The correlation between age of trees and degree of infestation by pathogen and pest was not observed. The significant genetic variability between two Aesculus species was revealed by the analysis of molecular variance (AMOVA) where both the intra- and the inter-species variation were found to be significant. It was shown that 73.0% of the genetic variance was contributed by the differentiation between A. hippocastanum and A.×carnea, whereas 27.0% was partitioned within species. The medium level of genetic diversity of Aesculus spp. was determined using SRAP and RAPD analyses. The mean value of genetic similarity was equal to 0.55 for common horse chestnut and 0.98 for red horse chestnut. Among 40 polymorphic SRAP and RAPD markers 17 were associated with degree of leaf damage caused by C. ohridella.

Did the parasitoid Pnigalio mediterraneus (Hymenoptera: Eulophidae) track the invasion of the horse chestnut leafminer?

How communities of natural enemies, such as parasitoids, adapt to the range expansion of their hosts or the arrival of a novel invasive host is an important question in invasion biology. Do parasitoids track the expansion of their hosts from their shared native range? Do they locally adapt both behaviorally and physiologically to the arrival of a novel species by shifting hosts? Few studies have addressed those questions, yet they are important to develop efficient methods to manage invasive species. Here we focus on Pnigalio mediterraneus Ferriere and Delucchi (Hymenoptera: Eulophidae), an important parasitoid of two major agricultural and ornamental pests, the olive fruit fly Bactrocera oleae Rossi (Diptera: Tephritidae) and the horse chestnut leafminer Cameraria ohridella Deschka & Dimic (Lepidoptera: Gracillariidae). C. ohridella recently invaded Europe starting from the Southern Balkans, whereas B. oleae has been associated since the Quaternary with wild olives in the Mediterranean, where it largely spread after the domestication of cultivated olives. We used two markers, the ribosomal spacer ITS2 and the mitochondrial gene COI. Although the ITS2 dataset provided little variation and no phylogeographic signal, analysis of mtDNA of 188 individuals of P. mediterraneus from 54 European localities allowed us to identify 53 haplotypes. Both nucleotide and haplotype diversity were higher for Mediterranean samples, and from samples reared from B. oleae. The statistical parsimony network identified one haplotype as the most frequent, ancestral and mainly associated with C. ohridella. Our findings suggest that P. mediterraneus locally host switched to C. ohridella from other hosts in the Balkans and later tracked the horse chestnut leafminer invasion over Europe. Therefore both host-tracking and ecological sorting could explain the current distribution of P. mediterraneus haplotypes.

Impulsive spatial control of invading pests by generalist predators

We model the conditions for pest eradication in a reaction-diffusion system made of a prey and a generalist predator through spatial impulsive control within a bounded domain. The motivating example is the control of the invasive horse chestnut leafminer moth through the yearly destruction of leaves in autumn, in which both the pest and its parasitoids overwinter. The model is made of two integro-partial differential equations, the integral portion describing the within-year immigration from the whole domain. The problem of pest eradication is strongly related to some appropriate eigenvalue problems. Basic properties of the principal eigenvalues of these problems are derived by using of Krein-Rutman's theorem and of comparison results for parabolic equations with non-local terms. Spatial control of the pest can be achieved, if one of these principal eigenvalues is large enough, at an exponential rate. This is true without and with parasitoids, the latter case being of course more rapid. We discuss the possible implementation of these results to the leafminer invasion problem and discuss complementary methods.

Oviposition patterns and larval damage by the invasive horse‐chestnut leaf miner Cameraria ohridella on different species of Aesculus

Abstract Preference–performance relationships are thought to be particularly important for sessile herbivores, such as leaf miners, whose choice of host plant is entirely determined by the ovipositing female. However, this relationship has seldom been examined between a non‐native herbivore and non‐native host plants. Leaf damage and oviposition patterns by the invasive horse‐chestnut leaf miner, Cameraria ohridella Deschka & Dimic, 1986 (Lepidoptera: Gracillariidae), were investigated on 11 different species of Aesculus L. (Sapindaceae) at the Royal Botanic Gardens, Kew. The tree species studied were of different continental origin and belonged to four different sections of the genus Aesculus. Oviposition was recorded on all the investigated species of Aesculus, with egg densities being greatest on Aesculus hippocastanum L. and Aesculus turbinata Blume. By contrast, mines were formed on only six out of the 11 studied species. There was a positive correlation between eggs laid on leaves of a species and levels of leaf tissue damage by C. ohridella, although eggs were laid on species on which larvae did not develop. Host suitability for C. ohridella was related to the phylogeny of the genus Aesculus. Species belonging to the section Aesculus were susceptible to the leaf miner; species in the section Pavia showed variable susceptibility; and species in the section Calothyrsus and Macrothyrsus were found to be resistant.

Population genetic characteristics of horse chestnut in Serbia

The general population genetic characteristics of cultivated horse chestnut trees excelling in growth, phenotype characteristics, type of inflorescence, productivity and resistance to the leafminer Cameraria ohridella Deschka and Dimic were analyzed in Serbia. The analyzed population genetic parameters point to fundamental differences in the genetic structure among the cultivated populations in Serbia. The study shows the variability in all properties among the populations and inter-individual variability within the populations. The variability and differential characteristics were assessed using statistical parameters, taking into account the satisfactory reflection of the hereditary potential. The assessed differences in the vitality and evolution potential of different populations can determine the methods of horse chestnut gene pool collection, reconstruction and improvement.

Lecanicillium Aphanocladii – A New Species to the Mycoflora of Lithuania and a New Pathogen of Tree Leaves Mining Insects

Abstract Pečiulytė D., Kačergius A., 2012: Lecanicillium aphanocladii - a new species to the mycoflora of Lithuania and a new pathogen of tree leaves mining insects [Lecanicillium aphanocladii - nauja Lietuvos mikofloros rūšis ir naujas medžių lapus minuojančių vabzdžių patogenas pasaulyje]. - Bot. Lith., 18(2); 133-146. Popular park and city trees horse chestnut, Aesculus hippocastanum L. and small-leaved linden, Tilia cordata Miller suffer from damages of leaf mining moths, Cameraria ohridella Deschka & Dimic 1968 and Lithocolletis issikii Kumata 1825 (= Phyllonorycter issikii Humber 1822 (both Lepidoptera, Gracillariidae), respectively. In 2010-2011, fungi causing moth caterpillar mortality under field conditions were investigated in Vilnius city, Lithuania. Statistical analyses showed high recovery percentage (30.36 % and 31.92 % from C. ohridella and L. issikii caterpillar cadavers, respectively) of a new fungus, which was thereafter identified as Lecanicillium aphanocladii Zare & W. Gams 2001 based on a morphological characteristics and ITS rDNR sequence, which showed 99 % identity with Aphanocladium aranearum (Petch) W. Gams 1971 GenBank accessions No. AF455489 and AF455405 of ribosomal RNA gene (now renamed as L. aphanockladii).

Digestive enzymes activity in subsequent generations of Cameraria ohridella larvae harvested from horse chestnut trees after treatment with imidacloprid

In the present study we describe the effect of chloronicotinoid pesticide (imidacloprid) on the digestive enzymes activity of the Cameraria ohridella larvae after lasting 1year sublethal exposure to imidacloprid pesticide. Caterpillars – L4 stage (fourth instar, hyperphagic tissue-feeding phase) – were collected from chemically protected white horse chestnut trees 1year after imidacloprid treatment, and compared with caterpillars collected from non-treated trees in a previous study. Enzymes activity of α-amylase, disaccharidases, glycosidases and proteases was assayed. The presence of pesticide in ingested food changed the digestive enzymes profile of caterpillars. The analysis of correlations between different digestive enzymes showed many significant correlations (P<0.05) among glycolytic activities like β-glucosidase and α-galactosidase activities. Statistically significant correlations for proteolytic activity were found between trypsin and chymotrypsin activity and aminopeptidase activity that occurred only in the 1st generation. PCA distinguished five primary components with eigenvalues higher than 1, from which the first two explain almost 59% of analyzed results. Surprisingly, in the pesticide treated groups significantly higher activities of sucrase and lactase in relation to control were found. In general, glycosidase (α-glucosidase, β-glucosidase and β-galactosidase) activities showed a similar pattern of activity in different generations. These results contrast with those obtained with control larvae, where significant differences in activities of α-glucosidase, β-glucosidase and β-galactosidase may result from the different quantity and quality food intake by subsequent generations of larvae. No inter-generation differences in total proteolytic activity were observed in treated larvae. The absolute value of total proteolytic activity was higher than that in the control group. The pesticide present in the vascular system of the horse chestnut tree significantly affected some of the digestive enzymes activities and – in consequence – also interrelationships between enzymes, what may affect the food digestion.