Molecular strategies of the pygmy grasshopper Eucriotettix oculatus adapting to long-term heavy metal pollution.

To study the heavy metal accumulation and its impact on insect exterior and chromosome morphology, and reveal the molecular mechanism of insects adapting to long-term heavy metal compound pollution habitats, this study, in the Diaojiang river basin, which has been polluted by heavy metals(HMs) for nearly a thousand years, two Eucriotettix oculatus populations was collected from mining and non-mining areas. It was found that the contents of 7 heavy metals (As, Cd, Pb, Zn, Cu, Sn, Sb) in E. oculatus of the mining area were higher than that in the non-mining 1-11 times. The analysis of morphology shows that the external morphology, the hind wing type and the chromosomal morphology of E. oculatus are significant differences between the two populations. Based on the heavy metal accumulation，morphological change, and stable population density, it is inferred that the mining area population has been affected by heavy metals and has adapted to the environment of heavy metals pollution. Then, by analyzing the transcriptome of the two populations, it was found that the digestion, immunity, excretion, endocrine, nerve, circulation, reproductive and other systems and lysosomes, endoplasmic reticulum and other cell structure-related gene expression were suppressed. This shows that the functions of the above-mentioned related systems of E. oculatus are inhibited by heavy metal stress. However, it has also been found that through the significant up-regulation of genes related to the above system, such as ATP2B, pepsin A, ubiquitin, AQP1, ACOX, ATPeV0A, SEC61A, CANX, ALDH7A1, DLD, aceE, Hsp40, and catalase, etc., and the down-regulation of MAPK signalling pathway genes, can enhanced nutrient absorption, improve energy metabolism, repair damaged cells and degrade abnormal proteins, maintain the stability of cells and systems, and resist heavy metal damage so that E. oculatus can adapt to the environment of heavy metal pollution for a long time.

A locust embryo as predictive developmental neurotoxicity testing system for pioneer axon pathway formation.

Exposure to environmental chemicals during in utero and early postnatal development can cause a wide range of neurological defects. Since current guidelines for identifying developmental neurotoxic chemicals depend on the use of large numbers of rodents in animal experiments, it has been proposed to design rapid and cost-efficient in vitro screening test batteries that are mainly based on mixed neuronal/glial cultures. However, cell culture tests do not assay correct wiring of neuronal circuits. The establishment of precise anatomical connectivity is a key event in the development of a functional brain. Here, we expose intact embryos of the locust (Locusta migratoria) in serum-free culture to test chemicals and visualize correct navigation of identified pioneer axons by fluorescence microscopy. We define separate toxicological endpoints for axonal elongation and navigation along a stereotyped pathway. To distinguish developmental neurotoxicity (DNT) from general toxicity, we quantify defects in axonal elongation and navigation in concentration-response curves and compare it to the biochemically determined viability of the embryo. The investigation of a panel of recognized DNT-positive and -negative test compounds supports a rather high predictability of this invertebrate embryo assay. Similar to the semaphorin-mediated guidance of neurites in mammalian cortex, correct axonal navigation of the locust pioneer axons relies on steering cues from members of this family of cell recognition molecules. Due to the evolutionary conserved mechanisms of neurite guidance, we suggest that our pioneer axon paradigm might provide mechanistically relevant information on the DNT potential of chemical agents on the processes of axon elongation, navigation, and fasciculation.

Scanning laser optical tomography resolves developmental neurotoxic effects on pioneer neurons.

Developmental neurotoxic compounds impair the developing human nervous system at lower doses than those affecting adults. Standardized test methods for assessing developmental neurotoxicity (DNT) require the use of high numbers of laboratory animals. Here, we use a novel assay that is based on the development of an intact insect embryo in serum-free culture. Neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons, extending axons along a well-defined pathway to the central nervous system. After exposure to test chemicals, we analyze pioneer neuron shape with conventional fluorescence microscopy and compare it to 3D images, obtained by scanning laser optical tomography (SLOT) and processed by a segmentation algorithm. The segmented SLOT images resolve the 3D structure of the pioneers, recognize pathfinding defects and are thus advantageous for detecting DNT-positive compounds. The defects in axon elongation and pathfinding of pioneer axons caused by two DNT-positive reference compounds (methylmercury chloride; sodium(meta)arsenite) are compared to the biochemically measured general viability of the embryo. Using conventional fluorescence microscopy to establish concentration-response curves of axon elongation, we show that this assay identifies methylmercury chloride and the pro-apoptotic compound staurosporine as developmental neurotoxicants.

Rhythmic change of adipokinetic hormones diurnally regulates locust vitellogenesis and egg development.

Adipokinetic hormones (AKHs), the neurohormones synthesized in the insect corpora cardiaca are known to mobilize lipids and carbohydrates for energy-consuming activities including reproduction. However, both inhibitory and stimulatory effects of AKHs on insect reproduction have been reported, and the underlying mechanisms remain elusive. Using the migratory locust, Locusta migratoria, as a model system, we report here that AKHs are expressed in response to rhythmic diel change, and AKH III expression increases markedly at photophase. Diurnal injection of AKH III but not AKH I or AKH II in adult females stimulates vitellogenesis and egg development. In contrast, AKH treatment at scotophase represses female reproduction. RNA interference-mediated knockdown of AKH receptor (AKHR) results in significantly reduced vitellogenin (Vg) expression in the fat body at photophase along with reduced Vg deposition in the ovary. AKHR knockdown also leads to decreased expression of Brummer, triacylglycerol lipase and trehalose transporter, accompanied by suppressed mobilization of triacylglycerol and trehalose. We propose that in addition to stimulating Vg expression at photophase, AKH/AKHR signalling is likely to regulate ovarian uptake of Vg via triacylglycerol mobilization and trehalose homeostasis. This study provides new insights into the understanding of AKH/AKHR signalling in the regulation of insect reproduction.

Effects of FABP knockdown on flight performance of the desert locust, Schistocerca gregaria.

During migratory flight, desert locusts rely on fatty acids as their predominant source of energy. Lipids mobilized in the fat body are transported to the flight muscles and enter the muscle cells as free fatty acids. It has been postulated that muscle fatty acid binding protein (FABP) is needed for the efficient translocation of fatty acids through the aqueous cytosol towards mitochondrial ß-oxidation. To assess whether FABP is required for this process, dsRNA was injected into freshly emerged adult males to knock down the expression of FABP. Three weeks after injection, FABP and its mRNA were undetectable in flight muscle, indicating efficient silencing of FABP expression. At rest, control and treated animals exhibited no morphological or behavioral differences. In tethered flight experiments, both control and treated insects were able to fly continually in the initial, carbohydrate-fueled phase of flight, and in both groups, lipids were mobilized and released into the hemolymph. Flight periods exceeding 30 min, however, when fatty acids become the main energy source, were rarely possible for FABP-depleted animals, while control insects continued to fly for more than 2 h. These results demonstrate that FABP is an essential element of skeletal muscle energy metabolism in vivo.

Aryl hydrocarbon receptor regulates the expression of LmGSTd7 and is associated with chlorpyrifos susceptibility in Locusta migratoria.

BACKGROUND: The aryl hydrocarbon receptor (AhR) belongs to the bHLH-PAS (basic Helix-Loop-Helix - Period/ARNT/Single minded) family of transcription factors. AhR is a ligand-activated transcription factor, which participates in the sensing and transmitting stimuli of endogenous and exogenous chemicals, and subsequently activates the transcription of genes related to various physiological and detoxification functions. RESULT: In this study, a single full-length LmAhR sequence was cloned and characterized. RNA interference (RNAi) and insecticide bioassays showed that LmAhR plays a vital role in chlorpyrifos susceptibility. To better identify aryl hydrocarbon receptor from locusta migratoria (LmAhR)-regulated genes involved in chlorpyrifos susceptibility, a comparative transcriptome analysis was performed using double-stranded (ds)GFP- and dsLmAhR-injected Locusta migratoria. Differential gene expression analysis identified 145 down-regulated and 67 up-regulated genes (P ≤ 0.05 and fold change ≥2) in dsLmAhR-knockdown insects. We selected 27 down-regulated genes and verified their expression levels using reverse transcription quantitative PCR. Finally, one glutathione S-transferase (GST) gene (LmGSTd7) was selected as a candidate detoxification gene and was further validated via RNAi and chlorpyrifos bioassays. CONCLUSION: Our data suggest that AhR is associated with chlorpyrifos susceptibility via the regulation of LmGSTd7 expression in L. migratoria. © 2019 Society of Chemical Industry.

An intact insect embryo for developmental neurotoxicity testing of directed axonal elongation.

Developmental neurotoxicity (DNT) of chemicals poses a serious threat to human health worldwide. Current in vivo test methods for assessing DNT require the use of high numbers of laboratory animals. Most alternative in vitro testing methods monitor rather simple toxicological endpoints, whereas the formation of a functional brain requires precisely timed navigation of axons within a complex tissue environment. We address this complexity by monitoring defects in axonal navigation of pioneer axons of intact locust embryos after exposure to chemicals. Embryos develop in serum-free culture with test chemicals, followed by immunolabeling of pioneer neurons. Defects in axon elongation of pioneer axons are quantified in concentration-response curves and compared to the general viability of the embryo, as measured by a resazurin assay. We show that selected chemical compounds interfering with calcium signaling, the cytoskeletal organization, and the reference developmental neurotoxicant rotenone, can be classified as DNT positive. The pesticide rotenone inhibits pioneer neuron elongation with a lower IC50 than the viability assay. The rho kinase inhibitor Y27632 can partially rescue outgrowth inhibition, supporting the classification of rotenone as a specific DNT positive compound. Since mechanisms of axonal guidance, such as growth cone navigation along molecular semaphorin gradients are conserved between locust and mammalian nervous systems, we will further explore the potential of this invertebrate preparation as an assay for testing the DNT potential of chemicals in humans.

Jaburetox, a natural insecticide derived from Jack Bean Urease, activates voltage-gated sodium channels to modulate insect behavior.

Jaburetox (Jbtx) is an insecticidal peptide derived from Canavalia ensiformis urease, whose mechanism of action is not completely elucidated. We employed behavioral, electromyographical and electrophysiological protocols to identify the cellular and molecular targets involved in the Jbtx entomotoxicity in cockroaches and locusts. In Nauphoeta cinerea, Jbtx (32 µg/g) altered the locomotory behaviour inducing a significative decrease in the distance travelled followed by a significant increase in stopped time (52 ±â€¯85 cm and 2573 ±â€¯89 s, p < .05, n = 40). Jbtx (8 to 32 µg/g body weight, respectively) also increased the leg and antennae grooming activities (p < .05, n = 40, respectively). Jbtx (8 to 16 µg/g) induced a maximum neuromuscular blockade of 80.72% (n = 6, p < .05) and was cardiotoxic, decreasing the cockroach heart rate. The electrophysiological profiles of both muscle and nerve of L. migratoria showed that Jbtx (2.5 × 10-7 and 2.5 × 10-3 µg/ body weight) induced a significant increase in the amplitude of nerve action potentials (n = 5, p < .05). Voltage clamp analysis of Jbtx (200 nM) applied in Xenopus laevis oocytes heterologously expressed with Nav 1.1 channels showed a significant increase in the sodium currents. In conclusion, this work revealed that the entomotoxic activity of Jbtx involves complex behavioral alterations that begins with an initial activation of voltage-gated sodium channels.

Antioxidant enzyme activity in responses to environmentally induced oxidative stress in the 5th instar nymphs of Aiolopus thalassinus (Orthoptera: Acrididae).

The response of antioxidant enzymes to oxidative environmental stress was determined in 5th instar nymphs of Aiolopus thalassinus (Orthoptera: Acrididae) collected from sites with different level of pollution with heavy metals, PO43-, and SO42-. The high polluted site induced higher DNA damage to individuals compared to the control site. The highest values of tail length (TL), tail moment (TM), and percent of DNA in tail (TDNA) were found in the gut of 5th instar nymphs from a high polluted site. Also, protein carbonyls and lipid peroxide levels were significantly higher in insects collected from polluted sites compared to those from the control site. A strong positive correlation between both protein carbonyl and lipid peroxide concentration and the pollution level of the sites was found in all tissues of the insects. The activity of superoxide dismutase (SOD) in the brain of insects collected from the high polluted site was significantly higher than that in the thoracic muscles and gut. We observed strong inhibition of catalase (CAT) activity. This effect was apparently caused by pollutants present at the high polluted site. The level of pollution significantly influenced polyphenol oxidase (PPO) activity in A. thalassinus nymphs in all examined tissues. The highest values were observed in the brain. The relationship between pollution and ascorbate peroxidase (APOX) activity in the examined tissues had no clear tendency. However, the lowest APOX activity was observed in individuals from the low polluted site. Level of pollution of sampling sites, oxidative stress biomarkers, and enzymatic response in A. thalanthsis 5th instar were negatively or positively correlated. Oxidative damage parameters, especially the percent of severed cells, lipid peroxides, and the activity of APOX, can be perceived as good markers of environmental multistress.

Enantioselective Synthesis and Activity of All Diastereoisomers of ( E)-Phytal, a Pheromone Component of the Moroccan Locust, Dociostaurus maroccanus.

The Moroccan locust, Dociostaurus maroccanus (Thunberg, 1815) (Orthoptera: Acrididae), is a polyphagous pest capable of inflicting large losses in agriculture under favorable environmental and climatic conditions. Currently, control of the pest relies solely on the application of conventional insecticides that have negative effects on the environment and human safety. In the search for a more rational, environmentally acceptable approach for locust control, we have previously reported that ( Z/ E)-phytal (1) is a male-produced candidate sex pheromone of this acridid. This molecule, with two stereogenic centers at C-7 and C-11, has four different diastereomers along with the Z/ E stereochemistry of the double bond at C-2. In this paper, we present for the first time the enantioselective synthesis of the four diastereomers of ( E)-phytal and their electrophysiological and behavioral activity on males and females. Our results demonstrate that the ( R, R)-phytal is the most active diastereomer in both assays, significantly attracting females in a double-choice Y olfactometer, and confirming the previous chromatographic assignment as component of the sex pheromone of the Moroccan locust.

The microRNAs let-7 and miR-278 regulate insect metamorphosis and oogenesis by targeting the juvenile hormone early-response gene Krüppel-homolog 1.

Krüppel-homolog 1 (Kr-h1), a zinc-finger transcription factor, inhibits larval metamorphosis and promotes adult reproduction by transducing juvenile hormone (JH). Although the transcriptional regulation of Kr-h1 has been extensively studied, little is known about its regulation at the post-transcriptional level. Using the migratory locust Locusta migratoria as a model system, we report here that the microRNAs let-7 and miR-278 bound to the Kr-h1 coding sequence and downregulated its expression. Application of let-7 and miR-278 mimics (agomiRs) significantly reduced the level of Kr-h1 transcripts, resulting in partially precocious metamorphosis in nymphs as well as markedly decreased yolk protein precursors, arrested ovarian development and blocked oocyte maturation in adults. Moreover, the expression of let-7 and miR-278 was repressed by JH, constituting a regulatory loop of JH signaling. This study thus reveals a previously unknown regulatory mechanism whereby JH suppresses the expression of let-7 and miR-278, which, together with JH induction of Kr-h1 transcription, prevents the precocious metamorphosis of nymphs and stimulates the reproduction of adult females. These results advance our understanding of the coordination of JH and miRNA regulation in insect development.

Pre-synaptic Muscarinic Excitation Enhances the Discrimination of Looming Stimuli in a Collision-Detection Neuron.

Visual neurons that track objects on a collision course are often finely tuned to their target stimuli because this is critical for survival. The presynaptic neural networks converging on these neurons and their role in tuning them remain poorly understood. We took advantage of well-known characteristics of one such neuron in the grasshopper visual system to investigate the properties of its presynaptic input network. We find the structure more complex than hitherto realized. In addition to dynamic lateral inhibition used to filter out background motion, presynaptic circuits include normalizing inhibition and excitatory interactions mediated by muscarinic acetylcholine receptors. These interactions preferentially boost responses to coherently expanding visual stimuli generated by colliding objects, as opposed to spatially incoherent controls, helping to discriminate between them. Hence, in addition to active dendritic conductances within collision-detecting neurons, multiple layers of inhibitory and excitatory presynaptic connections are needed to finely tune neural circuits for collision detection.

Evaluation of Drug Exposure and Metabolism in Locust and Zebrafish Brains Using Mass Spectrometry Imaging.

Studying how and where drugs are metabolized in the brain is challenging. In an entire organism, peripheral metabolism produces many of the same metabolites as those in the brain, and many of these metabolites can cross the blood-brain barrier from the periphery, thus making the relative contributions of hepatic and brain metabolism difficult to study in vivo. In addition, drugs and metabolites contained in ventricles and in the residual blood of capillaries in the brain may overestimate drugs' and metabolites' concentrations in the brain. In this study, we examine locusts and zebrafish using matrix assisted laser desorption ionization mass spectrometry imaging to study brain metabolism and distribution. These animal models are cost-effective and ethically sound for initial drug development studies.

RNAi-mediated knockdown of SPOOK reduces ecdysteroid titers and causes precocious metamorphosis in the desert locust Schistocerca gregaria.

The Halloween gene SPOOK (SPO) is involved in the production of the active metabolite of ecdysteroid, 20-hydroxyecdysone (20E), in insects. A previous study showed that RNAi-mediated knockdown of SPO in Schistocerca gregaria last instar nymphs markedly reduced the hemolymph 20E titer, but did not affect metamorphosis. In the present study, the effects of SPO interference on development were re-examined in this locust. Injections of SPO double-stranded RNA (dsSPO) into nymphs at mid and late instars significantly delayed nymphal development and interfered with molting. The 20E levels of dsSPO-treated nymphs were generally low, with a delayed, small peak, suggesting that disturbance of the 20E levels caused the above developmental abnormalities. A small proportion of the dsSPO-injected nymphs metamorphosed precociously, producing adults and adultoids. Precocious adults were characterized by small body size, short wings with abbreviated venation, and normal reproductive activity. Fourth instar nymphs that precociously metamorphosed at the following instar exhibited temporal expression patterns of ecdysone-induced protein 93F and the juvenile hormone (JH) early-inducible gene Krüppel homolog 1 similar to those observed at the last instar in normal nymphs. Adultoids displayed mating behavior and adultoid females developed eggs, but never laid eggs. JH injection around the expected time of the 20E peak in the dsSPO-injected nymphs completely inhibited the appearance of adultoids, suggesting that appearance of adultoids might be due to a reduced titer of JH rather than of 20E. These results suggest that SPO plays an important role in controlling morphogenesis, metamorphosis, and reproduction in S. gregaria.

Two Olfactory Pathways to Detect Aldehydes on Locust Mouthpart.

Sensing chemical cues is crucial for insects through their olfactory systems to adapt the environments. The receptors employed in insect olfactory system belong to the Odorant Receptor (ORs) and Ionotropic Receptor (IRs) families. In general, ORs and IRs are present in distinct olfactory sensory neurons and function independently. Here, we present evidence that in locust, the abundant host plant odor Hexanal is detected by both IR- and OR-expressing neurons. Use of the palp opening response (POR) as a simple behavioral paradigm in conjunction with RNA interference (RNAi) revealed that these two pathways are both needed for the detection of Hexanal. Two-color fluorescence in situ hybridization showed that OR2 and odorant-binding protein 1 (obp1) were co-localized in palps sensilla basiconica. Obp2a and IR8a were co-localized as well, but associated with sensilla chaetica on the palps. Furthermore, both OR2- and obp1-knockdowns showed reduced POR responses to Hexanal and E-2-Hexenal, and the same was true for Hexanal with IR8a- and obp2a-knockdowns. Detection to E-2-Hexenal was independent of IR8a-mediated gene silencing. Besides, Hexanal and E-2-Hexenal evoked dose-dependent responses in palp basiconica via extracellular recordings. Our results indicate that both OR and IR pathways are involved in the detection of one aldehyde.

Afidopyropen: New and potent modulator of insect transient receptor potential channels.

The commercial insecticides pymetrozine and pyrifluquinazon control plant-sucking pests by disturbing their coordination and ability to feed. We have previously shown that these compounds act by overstimulating and eventually silencing vanilloid-type transient receptor potential (TRPV) channels, which consist of two proteins, Nanchung and Inactive, that are co-expressed exclusively in insect chordotonal stretch receptor neurons. Here we show that a new insecticidal compound, afidopyropen, modulates chordotonal organs of American grasshoppers (Schistocerca americana) in the same fashion. Afidopyropen stimulated heterologously expressed TRPV channels from two different insect species - fruit fly (Drosophila melanogaster) and pea aphid (Acyrthosiphon pisum) - but did not affect function of the mammalian TRPV channel TRPV4. Activation of the insect TRPVs required simultaneous expression of both Nanchung and Inactive proteins. Tritium-labeled afidopyropen bound fruit fly TRPVs with higher affinity than pymetrozine and competed with pymetrozine for binding. Nanchung protein formed the main binding interface for afidopyropen, whereas co-expression of Inactive dramatically increased binding affinity. Another modulator of chordotonal organs, flonicamid, did not activate insect TRPV channels, nor did it compete with afidopyropen for binding, indicating that it has a different target site. These results define afidopyropen as a new, potent and specific modulator of insect TRPV channels, and provide insight into the unique binding mode of these compounds.

Assessment of oxidative stress and activities of antioxidant enzymes depicts the negative systemic effect of iron-containing fertilizers and plant phenolic compounds in the desert locust.

For herbivore insects, digesting can be somewhat challenging, as the defense mechanisms evolved by plants, including the release of phenolics like the non-protein amino acid L-3,4-dihydroxyphenylalanine (L-DOPA), can cause fitness costs. In addition, industrial and agricultural activities have elevated the amounts of iron that can be found in nature and more particularly FeSO4 that is used as fertilizer. Traces of iron can enhance the auto-oxidation of L-DOPA, in turn, generating reactive oxygen species (ROS) and consequently oxidative stress in insects. We examined the effects of the ion Fe2+ (as FeSO4) and L-DOPA on fifth instars of the desert locust Schistocerca gregaria. We measured the level of oxidative damage occurring to macromolecules (proteins and lipids) from midgut and thoracic tissues and assessed the activities of responsive antioxidant enzymes. Injected L-DOPA and redox-active metal iron generated ROS which caused oxidative damages to proteins and lipids to S. gregaria. The protein carbonyls and lipid peroxides present in tissue homogenates were elevated in treated insects. No synergism was observed when L-DOPA was co-injected with Fe2+. K m values of superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) were 4.3, 2.6, and 4.0 mM in thoracic muscles and 5.00, 2.43, and 1.66 mM in whole midgut for SOD, GR, and GPx, respectively, and 8.3 and 3.43 M for catalase (CAT) in the two tissues, respectively. These results suggest higher affinities of GPx and CAT to H2O2 in midgut than in muscles. The time-course changes in activities of antioxidant enzymes and amounts of protein carbonyls and lipid peroxides showed fluctuating patterns, suggesting complex interactions among macromolecules, L-DOPA and FeSO4, and their degradation products. Our results demonstrated the stressful effects of L-DOPA and FeSO4, proving that iron-containing fertilizers are pollutants that can strongly affect S. gregaria.

Susceptibility and potential biochemical mechanism of Oedaleus asiaticus to beta-cypermethrin and deltamethrin in the Inner Mongolia, China.

Oedaleus asiaticus is a highly destructive grass pest in Inner Mongolia, China, and likely developed resistance to pyrethroid insecticides due to their frequent application for control of this locust. In this study, the susceptibility of five field populations of O. asiaticus to two pyrethroid insecticides was investigated. The Wulate Middle Banner (WB) population was the least susceptible, whereas the Ewenki Banner (EB) population appeared to be the most sensitive. The WB population was 3.16 and 5.15-fold less sensitive to beta-cypermethrin and deltamethrin than EB population, respectively. Further, the enzyme activities and mRNA expression levels of carboxylesterase (CarE) and glutathione-S-transferase (GST) were determined and we found that their activities in the WB population were 5.15 and 2.8-fold higher than those in the EB population, respectively. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that the mRNA expression levels of CarE and GST genes were positively correlated with the LD50 in the WB, Siziwang Banner (SB) and EB populations. Our findings suggest that differences in susceptibility to pyrethroids in O. asiaticus might be attributed to the elevated activities and mRNA expression levels of CarE and GST genes.

The level of DNA damage in adult grasshoppers Chorthippus biguttulus (Orthoptera, Acrididae) following dimethoate exposure is dependent on the insects' habitat.

The comet assay was used to study the DNA damage that was induced by dimethoate in the hemocyte cells of adult Chorthippus biguttulus grasshoppers (Insecta: Orthoptera) that originated from two sites with varying levels of pollution. The primary focus of the study was to examine whether continuous exposure to environmental stress can modify the effect of pesticides on genome stability. After three days of acclimation to laboratory conditions, the level of DNA damage in the hemocytes of Bow-winged grasshoppers was within a similar range in the insects from both areas. However, the level of DNA damage following dimethoate treatment was significantly higher in the insects from the reference area (Pogoria) than in the individuals from the heavily polluted location (Szopienice). Four hours after pesticide treatment, the Tail DNA (TDNA) in the hemocytes of the male and female specimens from Pogoria was as high as 75% and 50% respectively, whereas the values in males and females from Szopienice only reached 30% and 20%, respectively. A rapid decrease in DNA damage was observed in both populations 24 h after the pesticide application. The habitat of an insect (site), the administration of the dimethoate (treatment), and the period following the application of the pesticide (time), all significantly influenced the levels of DNA damage. No interactions related to TDNA were observed between the variables 'sex' and 'treatment'. Similarly, the variable 'sex', when analyzed alongside 'treatment' and 'site' (the area from which the insects were collected), or 'treatment' and 'time' had no influence on TL. Exposure to dimethoate undoubtedly contributed to the formation of DNA damage in the hemocytes of adult C. biguttulus. However, the level of damage was clearly dependent on the place where the insects were captured.