Parental experiences orchestrate locust egg hatching synchrony by regulating nuclear export of precursor miRNA.

Parental experiences can affect the phenotypic plasticity of offspring. In locusts, the population density that adults experience regulates the number and hatching synchrony of their eggs, contributing to locust outbreaks. However, the pathway of signal transmission from parents to offspring remains unclear. Here, we find that transcription factor Forkhead box protein N1 (FOXN1) responds to high population density and activates the polypyrimidine tract-binding protein 1 (Ptbp1) in locusts. FOXN1-PTBP1 serves as an upstream regulator of miR-276, a miRNA to control egg-hatching synchrony. PTBP1 boosts the nucleo-cytoplasmic transport of pre-miR-276 in a "CU motif"-dependent manner, by collaborating with the primary exportin protein exportin 5 (XPO5). Enhanced nuclear export of pre-miR-276 elevates miR-276 expression in terminal oocytes, where FOXN1 activates Ptbp1 and leads to egg-hatching synchrony in response to high population density. Additionally, PTBP1-prompted nuclear export of pre-miR-276 is conserved in insects, implying a ubiquitous mechanism to mediate transgenerational effects.

A neural m6A pathway regulates behavioral aggregation in migratory locusts.

RNA N6-methyladenosine (m6A), as the most abundant modification of messenger RNA, can modulate insect behaviors, but its specific roles in aggregation behaviors remain unexplored. Here, we conducted a comprehensive molecular and physiological characterization of the individual components of the methyltransferase and demethylase in the migratory locust Locusta migratoria. Our results demonstrated that METTL3, METTL14 and ALKBH5 were dominantly expressed in the brain and exhibited remarkable responses to crowding or isolation. The individual knockdown of methyltransferases (i.e., METTL3 and METTL14) promoted locust movement and conspecific attraction, whereas ALKBH5 knockdown induced a behavioral shift toward the solitary phase. Furthermore, global transcriptome profiles revealed that m6A modification could regulate the orchestration of gene expression to fine tune the behavioral aggregation of locusts. In summary, our in vivo characterization of the m6A functions in migratory locusts clearly demonstrated the crucial roles of the m6A pathway in effectively modulating aggregation behaviors.

PLIN2-induced ectopic lipid accumulation promotes muscle ageing in gregarious locusts.

Ageing plasticity represents the flexibility of the ageing process in response to non-genetic factors, occurring commonly in animals. However, the regulatory mechanisms underlying ageing plasticity are largely unclear. The density-dependent polyphenism of locusts, Locusta migratoria, displays dramatic lifespan divergence between solitary and gregarious phases, providing a useful system for studying ageing plasticity. Here, we found that gregarious locusts displayed faster locomotor deficits and increased muscle degeneration on ageing than solitary locusts. Comparative transcriptome analysis in flight muscles revealed significant differences in transcriptional patterns on ageing between two phases. RNA interference screening showed that the knockdown of the upregulated PLIN2 gene significantly relieved the ageing-related flight impairments in gregarious locusts. Mechanistically, the gradual upregulation of PLIN2 could induce the accumulation of ectopic lipid droplets and triacylglycerols in flight muscles during the ageing process. Further experiments suggested that ectopic lipid accumulation led to an ageing-related ß-oxidation decline through limiting fatty acid transport and content. These findings reveal the key roles of lipid metabolism in the differences of muscle ageing between solitary and gregarious locusts and provide a potential mechanism underlying environment-induced muscle ageing plasticity.

New estimates of genome size in Orthoptera and their evolutionary implications.

Animal genomes vary widely in size, and much of their architecture and content remains poorly understood. Even among related groups, such as orders of insects, genomes may vary in size by orders of magnitude-for reasons unknown. The largest known insect genomes were repeatedly found in Orthoptera, e.g., Podisma pedestris (1C = 16.93 pg), Stethophyma grossum (1C = 18.48 pg) and Bryodemella holdereri (1C = 18.64 pg). While all these species belong to the suborder of Caelifera, the ensiferan Deracantha onos (1C = 19.60 pg) was recently found to have the largest genome. Here, we present new genome size estimates of 50 further species of Ensifera (superfamilies Gryllidea, Tettigoniidea) and Caelifera (Acrididae, Tetrigidae) based on flow cytometric measurements. We found that Bryodemella tuberculata (Caelifera: Acrididae) has the so far largest measured genome of all insects with 1C = 21.96 pg (21.48 gBp). Species of Orthoptera with 2n = 16 and 2n = 22 chromosomes have significantly larger genomes than species with other chromosome counts. Gryllidea genomes vary between 1C = 0.95 and 2.88 pg, and Tetrigidae between 1C = 2.18 and 2.41, while the genomes of all other studied Orthoptera range in size from 1C = 1.37 to 21.96 pg. Reconstructing ancestral genome sizes based on a phylogenetic tree of mitochondrial genomic data, we found genome size values of >15.84 pg only for the nodes of Bryodemella holdereri / B. tuberculata and Chrysochraon dispar / Euthystira brachyptera. The predicted values of ancestral genome sizes are 6.19 pg for Orthoptera, 5.37 pg for Ensifera, and 7.28 pg for Caelifera. The reasons for the large genomes in Orthoptera remain largely unknown, but a duplication or polyploidization seems unlikely as chromosome numbers do not differ much. Sequence-based genomic studies may shed light on the underlying evolutionary mechanisms.

Mwhitiwhiti Aotearoa: Phylogeny and synonymy of the silent alpine grasshopper radiation of New Zealand (Orthoptera: Acrididae).

Aotearoa New Zealand has a fauna of endemic alpine grasshoppers, consisting of thirteen species distributed among four genera. The many re-classifications of species within this group and the presence of species complexes highlight the uncertainty that surrounds relationships within and between these genera. High-throughput Next Generation Sequencing was used to assemble the complete mitochondrial genomes, 45S ribosomal cassettes and histone sequences of New Zealands four endemic alpine genera: Alpinacris, Brachaspis, Paprides and Sigaus. Phylogenetic analysis of these molecular datasets, as individual genes, partitions and combinations returned a consistent topology that is incompatible with the current classification. The genera Sigaus, Alpinacris, and Paprides all exhibit paraphyly. A consideration of the pronotum, epiphallus and terminalia of adult specimens reveals species-specific differences, but fails to provide compelling evidence for species groups justifying distinct genera. In combination with phylogenetic, morphological and spatial evidence we propose a simplified taxonomy consisting of a single genus for the mwhitiwhiti Aotearoa species radiation.

Sexual repurposing of juvenile aposematism in locusts.

Adaptive plasticity requires an integrated suite of functional responses to environmental variation, which can include social communication across life stages. Desert locusts (Schistocerca gregaria) exhibit an extreme example of phenotypic plasticity called phase polyphenism, in which a suite of behavioral and morphological traits differ according to local population density. Male and female juveniles developing at low population densities exhibit green- or sand-colored background-matching camouflage, while at high densities they show contrasting yellow and black aposematic patterning that deters predators. The predominant background colors of these phenotypes (green/sand/yellow) all depend on expression of the carotenoid-binding "Yellow Protein" (YP). Gregarious (high-density) adults of both sexes are initially pinkish, before a YP-mediated yellowing reoccurs upon sexual maturation. Yellow color is especially prominent in gregarious males, but the reason for this difference has been unknown since phase polyphenism was first described in 1921. Here, we use RNA interference to show that gregarious male yellowing acts as an intrasexual warning signal, which forms a multimodal signal with the antiaphrodisiac pheromone phenylacetonitrile (PAN) to prevent mistaken sexual harassment from other males during scramble mating in a swarm. Socially mediated reexpression of YP thus adaptively repurposes a juvenile signal that deters predators into an adult signal that deters undesirable mates. These findings reveal a previously underappreciated sexual dimension to locust phase polyphenism, and promote locusts as a model for investigating the relative contributions of natural versus sexual selection in the evolution of phenotypic plasticity.

Genomic evidence of speciation by fusion in a recent radiation of grasshoppers.

Postdivergence gene flow can trigger a number of creative evolutionary outcomes, ranging from the transfer of beneficial alleles across species boundaries (i.e., adaptive introgression) to the formation of new species (i.e., hybrid speciation). Although neutral and adaptive introgression has been broadly documented in nature, hybrid speciation is assumed to be rare and the evolutionary and ecological context facilitating this phenomenon still remains controversial. Through combining genomic and phenotypic data, we evaluate the hypothesis that the dual feeding regime (based on both scrub legumes and gramineous herbs) of the taxonomically controversial grasshopper Chorthippus saulcyi algoaldensis resulted from hybridization between the sister taxa C. binotatus (that exclusively feeds on scrub legumes) and C. saulcyi (that only feeds on gramineous herbs). Genetic clustering analyses and inferences from coalescent-based demographic simulations confirm that C. s. algoaldensis represents an independently evolving lineage and support the ancient hybrid origin of this taxon (about 1.4 Ma), which sheds light on its uncertain phylogenetic position and might explain its broader trophic niche. We propose a Pleistocene hybrid speciation model where range shifts resulting from climatic oscillations can promote the formation of hybrid swarms and facilitate their long-term persistence through geographic isolation from parental forms in topographically complex landscapes.

Transcriptomic data reveals nuclear-mitochondrial discordance in Gomphocerinae grasshoppers (Insecta: Orthoptera: Acrididae).

The phylogeny of many groups of Orthoptera remains poorly understood. Previous phylogenetic studies largely restricted to few mitochondrial markers found many species in the grasshopper subfamily Gomphocerinae to be para- or polyphyletic, presumably because of incomplete lineage sorting and ongoing hybridization between putatively young lineages. Resolving the phylogeny of the Chorthippus biguttulus species complex is important because many morphologically cryptic species occupy overlapping ranges across Eurasia and serve important ecological functions. We investigated whether multispecies coalescent analysis of 540 genes generated by transcriptome sequencing could resolve the phylogeny of the C. biguttulus complex and related Gomphocerinae species. Our divergence time estimates confirm that Gomphocerinae is a very young radiation, with an age estimated at 1.38 (2.35-0.77) mya for the C. biguttulus complex. Our estimated topology based on complete mitogenomes recovered some species as para- or polyphyletic. In contrast, the multispecies coalescent based on nuclear genes retrieved all species as monophyletic clusters, corroborating most taxonomic hypotheses. Our results underline the importance of using nuclear multispecies coalescent methods for studying young radiations and highlight the need of further taxonomic revision in Gomphocerinae grasshoppers.

Prothoracicostatic Activity of the Ecdysis-Regulating Neuropeptide Crustacean Cardioactive Peptide (CCAP) in the Desert Locust.

Accurate control of innate behaviors associated with developmental transitions requires functional integration of hormonal and neural signals. Insect molting is regulated by a set of neuropeptides, which trigger periodic pulses in ecdysteroid hormone titers and coordinate shedding of the old cuticle during ecdysis. In the current study, we demonstrate that crustacean cardioactive peptide (CCAP), a structurally conserved neuropeptide described to induce the ecdysis motor program, also exhibits a previously unknown prothoracicostatic activity to regulate ecdysteroid production in the desert locust, Schistocerca gregaria. We identified the locust genes encoding the CCAP precursor and three G protein-coupled receptors that are activated by CCAP with EC50 values in the (sub)nanomolar range. Spatiotemporal expression profiles of the receptors revealed expression in the prothoracic glands, the endocrine organs where ecdysteroidogenesis occurs. RNAi-mediated knockdown of CCAP precursor or receptors resulted in significantly elevated transcript levels of several Halloween genes, which encode ecdysteroid biosynthesis enzymes, and in elevated ecdysteroid levels one day prior to ecdysis. Moreover, prothoracic gland explants exhibited decreased secretion of ecdysteroids in the presence of CCAP. Our results unequivocally identify CCAP as the first prothoracicostatic peptide discovered in a hemimetabolan species and reveal the existence of an intricate interplay between CCAP signaling and ecdysteroidogenesis.

One hundred years of phase polymorphism research in locusts.

One hundred years ago in 1921, Sir Boris Uvarov recognized that two locust species are one species but appearing in two different phases, a solitarious and a gregarious phase. As locust swarms are still a big problem affecting millions of people, basic research has tried to understand the causes for the transition between phases. This phenomenon of phase polymorphism, now called polyphenism, is a very complex multifactorial process and this short review will draw attention to this important aspect of insect research.

Historical isolation facilitates species radiation by sexual selection: Insights from Chorthippus grasshoppers.

Theoretical and empirical studies have shown that species radiations are facilitated when a trait under divergent natural selection is also involved in sexual selection. It is yet unclear how quick and effective radiations are where assortative mating is unrelated to the ecological environment and primarily results from sexual selection. We address this question using sympatric grasshopper species of the genus Chorthippus, which have evolved strong behavioural isolation while lacking noticeable ecomorphological divergence. Mitochondrial genomes suggest that the radiation is relatively recent, dating to the mid-Pleistocene, which leads to extensive incomplete lineage sorting throughout the mitochondrial and nuclear genomes. Nuclear data shows that hybrids are absent in sympatric localities but that all species have experienced gene flow, confirming that reproductive isolation is strong but remains incomplete. Demographic modelling is most consistent with a long period of geographic isolation, followed by secondary contact and extensive introgression. Such initial periods of geographic isolation might facilitate the association between male signaling and female preference, permitting the coexistence of sympatric species that are genetically, morphologically, and ecologically similar, but otherwise behave mostly as good biological species.

Juvenile hormone acts through FoxO to promote Cdc2 and Orc5 transcription for polyploidy-dependent vitellogenesis.

Vitellogenin (Vg) is a prerequisite for egg production and embryonic development after ovipositioning in oviparous animals. In many insects, juvenile hormone (JH) promotes fat body cell polyploidization for the massive Vg synthesis required for the maturation of multiple oocytes, but the underlying mechanisms remain poorly understood. Using the migratory locust Locusta migratoria as a model system, we report here that JH induces the dephosphorylation of Forkhead box O transcription factor (FoxO) through a signaling cascade including leucine carboxyl methyltransferase 1 (LCMT1) and protein phosphatase 2A (PP2A). JH promotes PP2A activity via LCMT1-mediated methylation, consequently triggering FoxO dephosphorylation. Dephosphorylated FoxO binds to the upstream region of two endocycle-related genes, cell-division-cycle 2 (Cdc2) and origin-recognition-complex subunit 5 (Orc5), and activates their transcription. Depletion of FoxO, Cdc2 or Orc5 results in blocked polyploidization of fat body cells, accompanied by markedly reduced Vg expression, impaired oocyte maturation and arrested ovarian development. The results suggest that JH acts via LCMT1-PP2A-FoxO to regulate Cdc2 and Orc5 expression, and to enhance ploidy of fat body cells in preparation for the large-scale Vg synthesis required for synchronous maturation of multiple eggs.

An alpine grasshopper radiation older than the mountains, on Ka Tiritiri o te Moana (Southern Alps) of Aotearoa (New Zealand).

In New Zealand, 13 flightless species of endemic grasshopper are associated with alpine habitats and freeze tolerance. We examined the phylogenetic relationships of the New Zealand species and a subset of Australian alpine grasshoppers using DNA sequences from the entire mitochondrial genome, nuclear 45S rRNA and Histone H3 and H4 loci. Within our sampling, the New Zealand alpine taxa are monophyletic and sister to a pair of alpine Tasmanian grasshoppers. We used six Orthopteran fossils to calibrate a molecular clock analysis to infer that the most recent common ancestor of New Zealand and Tasmanian grasshoppers existed about 20 million years ago, before alpine habitat was available in New Zealand. We inferred a radiation of New Zealand grasshoppers ~13-15 Mya, suggesting alpine species diversification occurred in New Zealand well before the Southern Alps were formed by the mountain building events of the Kaikoura Orogeny 2-5 Mya. This would suggest that either the ancestors of today's New Zealand grasshoppers were not dependent on living in the alpine zone, or they diversified outside of New Zealand.

First draft genome assembly of the desert locust, Schistocerca gregaria.

Background: At the time of publication, the most devastating desert locust crisis in decades is affecting East Africa, the Arabian Peninsula and South-West Asia. The situation is extremely alarming in East Africa, where Kenya, Ethiopia and Somalia face an unprecedented threat to food security and livelihoods. Most of the time, however, locusts do not occur in swarms, but live as relatively harmless solitary insects. The phenotypically distinct solitarious and gregarious locust phases differ markedly in many aspects of behaviour, physiology and morphology, making them an excellent model to study how environmental factors shape behaviour and development. A better understanding of the extreme phenotypic plasticity in desert locusts will offer new, more environmentally sustainable ways of fighting devastating swarms. Methods: High molecular weight DNA derived from two adult males was used for Mate Pair and Paired End Illumina sequencing and PacBio sequencing. A reliable reference genome of Schistocerca gregaria was assembled using the ABySS pipeline, scaffolding was improved using LINKS. Results: In total, 1,316 Gb Illumina reads and 112 Gb PacBio reads were produced and assembled. The resulting draft genome consists of 8,817,834,205 bp organised in 955,015 scaffolds with an N50 of 157,705 bp, making the desert locust genome the largest insect genome sequenced and assembled to date. In total, 18,815 protein-encoding genes are predicted in the desert locust genome, of which 13,646 (72.53%) obtained at least one functional assignment based on similarity to known proteins. Conclusions: The desert locust genome data will contribute greatly to studies of phenotypic plasticity, physiology, neurobiology, molecular ecology, evolutionary genetics and comparative genomics, and will promote the desert locust's use as a model system. The data will also facilitate the development of novel, more sustainable strategies for preventing or combating swarms of these infamous insects.

New ways to acquire resistance: imperfect convergence in insect adaptations to a potent plant toxin.

Evolution of insensitivity to the toxic effects of cardiac glycosides has become a model in the study of convergent evolution, as five taxonomic orders of insects use the same few similar amino acid substitutions in the otherwise highly conserved Na,K-ATPase α. We show here that insensitivity in pyrgomorphid grasshoppers evolved along a slightly divergent path. As in other lineages, duplication of the Na,K-ATPase α gene paved the way for subfunctionalization: one copy maintains the ancestral, sensitive state, while the other copy is resistant. Nonetheless, in contrast with all other investigated insects, the grasshoppers' resistant copy shows length variation by two amino acids in the first extracellular loop, the main part of the cardiac glycoside-binding pocket. RT-qPCR analyses confirmed that this copy is predominantly expressed in tissues exposed to the toxins, while the ancestral copy predominates in the nervous tissue. Functional tests with genetically engineered Drosophila Na,K-ATPases bearing the first extracellular loop of the pyrgomorphid genes showed the derived form to be highly resistant, while the ancestral state is sensitive. Thus, we report convergence in gene duplication and in the gene targets for toxin insensitivity; however, the means to the phenotypic end have been novel in pyrgomorphid grasshoppers.

CREB-B acts as a key mediator of NPF/NO pathway involved in phase-related locomotor plasticity in locusts.

Gene expression changes in neural systems are essential for environment-induced behavioral plasticity in animals; however, neuronal signaling pathways mediating the effect of external stimuli on transcriptional changes are largely unknown. Recently, we have demonstrated that the neuropeptide F (NPF)/nitric oxide (NO) signaling pathway plays a regulatory role in phase-related locomotor plasticity in the migratory locust, Locusta migratoria. Here, we report that a conserved transcription factor, cAMP response element-binding protein B (CREB-B), is a key mediator involved in the signaling pathway from NPF2 to NOS in the migratory locust, triggering locomotor activity shift between solitarious and gregarious phases. We find that CREB-B directly activates brain NOS expression by interacting with NOS promoter region. The phosphorylation at serine 110 site of CREB-B dynamically changes in response to population density variation and is negatively controlled by NPF2. The involvement of CREB-B in NPF2-regulated locomotor plasticity is further validated by RNAi experiment and behavioral assay. Furthermore, we reveal that protein kinase A mediates the regulatory effects of NPF2 on CREB-B phosphorylation and NOS transcription. These findings highlight a precise signal cascade underlying environment-induced behavioral plasticity.

Role of PTP/PTK trans activated insulin-like signalling pathway in regulation of grasshopper (Oedaleus asiaticus) development.

Protein tyrosine phosphatase (PTPs) and protein tyrosine kinase (PTKs) genes are responsible for the regulation of insect insulin-like pathway (ILP), cells growth, metabolism initiation, gene transcription and observing immune response. Signal transduction in insect cell is also associated with PTPs and PTKs. The grasshopper (Oedaleus asiaticus) 'Bey-Bienko' were treated with dsRNA of protein tyrosine non-receptor type 4 (PTPN4) and protein tyrosine kinase 5 (PTK5) along with control (water). Applying dsPTK5 treatments in 5th instar of Oedaleus asiaticus, significant reduction was recorded in body dry mass, growth rate and overall performance except survival rate. Whereas with PTPN4, no such significant impact on all of these growth parameters was recorded. Expression of genes in ILP 5th instar of Oedaleus asiaticus by the application of dsPTPN4 and dsPTK5 revealed that PTK, INSR (insulin receptor), IRS (insulin receptor substrate), PI3K (phosphoinositide 3-kinase), PDK (3-phosphoinositide-dependent protein kinase), Akt (protein kinase B) and FOXO (forkhead transcription factor) significantly expressed with downregulation except PTPN4, which remained non-significant. On the other hand, the phosphorylation level of ILP four proteins in O. asiaticus with the treatment of dsPTPN4 and dsPTK5 significantly affected P-IRS and P-FOXO, while P-INSR and P-AKT remained stable at the probability level of 5%. This indicated that the stress response in the O. asiaticus insulin-like signalling pathway (ILP) reduced. Regarding association of protective enzymatic activities, ROS (relative oxygen species), CAT (catalase) and PO (phenol oxidase) increased significantly with exposure to dsPTK5 as compared to dsPTPN4 and control, while exposure of 5th instar of O. asiaticus to dsPTPN4 treatment slightly raised CAT and PO activities with but significant contribution. No such significant effect on MFO and POD was seen using dsPTPN4 and dsPTK5. This showed that in the ILP of O. asiaticus, PTK5 was detrimental to growth, body mass and overall performance, which ultimately benefited insect detoxification with high-energy cost.

A ß-carotene-binding protein carrying a red pigment regulates body-color transition between green and black in locusts.

Changes of body color have important effects for animals in adapting to variable environments. The migratory locust exhibits body color polyphenism between solitary and gregarious individuals, with the former displaying a uniform green coloration and the latter having a prominent pattern of black dorsal and brown ventral surface. However, the molecular mechanism underlying the density-dependent body color changes of conspecific locusts remain largely unknown. Here, we found that upregulation of ß-carotene-binding protein promotes the accumulation of red pigment, which added to the green color palette present in solitary locusts changes it from green to black, and that downregulation of this protein led to the reverse, changing the color of gregarious locusts from black to green. Our results provide insight that color changes of locusts are dependent on variation in the red ß-carotene pigment binding to ßCBP. This finding of animal coloration corresponds with trichromatic theory of color vision.

Juvenile hormone-dependent Kazal-type serine protease inhibitor Greglin safeguards insect vitellogenesis and egg production.

In addition to preventing insect metamorphosis, juvenile hormone (JH) is known to stimulate aspects of insect reproduction. However, the molecular mechanisms of JH action in insect reproduction remain largely unknown. By reanalyzing the transcriptomic data from adults and other developmental stages of the migratory locust Locusta migratoria, we identified a gene coding for Kazal-type protease inhibitor, previously named Greglin. Greglin is specifically expressed in adult females and most abundant in the fat body and ovaries. Interestingly, Greglin is among the top 3 of highly expressed genes in adult female locusts, after 2 vitellogenin ( Vg) genes. Greglin is induced by JH and expressed at remarkably high levels in the vitellogenic stage. Knockdown of Greglin in adult female locusts results in accelerated degradation of serine protease substrate and significantly reduced levels of Greglin protein in hemolymph and ovaries. The consequent phenotypes include blocked oocyte maturation, arrested ovarian growth and shrunken follicular epithelium, as well as declines in egg number and hatchability. The data provide the first evidence, to our knowledge, that JH-dependent Greglin is involved in locust vitellogenesis and oocyte maturation likely by protecting vitellogenesis and other forms of yolk precursors from proteolysis. The result offers new insights into the regulation of JH and function of protease inhibitors in insect vitellogenesis, oocyte maturation and fecundity.-Guo, W., Wu, Z., Yang, L., Cai, Z., Zhao, L., Zhou, S. Juvenile hormone-dependent Kazal-type serine protease inhibitor Greglin safeguards insect vitellogenesis and egg production.

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.