Insect Enzyme Research Articles

Scavenging H2O2 of plant host by saliva catalase of leafhopper vector benefits viral transmission.

Catalase (CAT) is the main reactive oxygen species (ROS)-scavenging enzyme in plants and insects. However, it remains elusive whether and how insect saliva CAT suppresses ROS-mediated plant defense, thereby promoting initial virus transmission by insect vectors. Here, we investigated how leafhopper Recilia dorsalis catalase (RdCAT) was secreted from insect salivary glands into rice phloem, and how it was perceived by rice chaperone NO CATALASE ACTIVITY1 (OsNCA1) to scavenge excessive H2O2 during insect-to-plant virus transmission. We found that the interaction of OsNCA1 with RdCAT activated its enzymatic activity to decompose H2O2 in rice plants during leafhopper feeding. However, initial insect feeding did not significantly change rice CATs transcripts. Knockout of OsNCA1 in transgenic lines decreased leafhopper feeding-activated CAT activity and caused higher H2O2 accumulation. A devastating rice reovirus activated RdCAT expression and promoted the cosecretion of virions and RdCAT into leafhopper salivary cavities and ultimately into the phloem. Virus-mediated increase of RdCAT secretion suppressed excessive H2O2, thereby promoting host attractiveness to insect vectors and initial virus transmission. Our findings provide insights into how insect saliva CAT is secreted and perceived by plant chaperones to suppress the early H2O2 burst during insect feeding, thereby facilitating viral transmission.

Purification and Characterization of Proteinaceous Thermostable α-Amylase Inhibitor from Sardinian Common Bean Nieddone Cultivar (Phaseolus vulgaris L.).

The increasing need for new treatments for obesity and diabetes has led to the development of new drugs and food supplements that could reduce carbohydrate absorption. Many starch blockers, based on common bean proteinaceous inhibitors against α-amylase (α-AI), are already present on the market. The extraction and purification of α-amylase inhibitor from a promising common bean cultivar from Sardinia (Nieddone) is described, highlighting the unique value of the Nieddone cultivar, particularly for its inhibitory activity on digestive enzymes and its complete lack of a hemagglutination effect on human red blood cells. The purification of α-AI involved two chromatographic steps (IEC and SEC) and was essential for revealing certain properties of the inhibitor. The purified inhibitor has a tetrameric structure (α2β2) and a molecular weight of approximately 42 kDa, as determined by SEC and SDS-PAGE, confirming it as a lectin-like inhibitor. The identification of the α-AI sequence was obtained by bottom-up high-resolution mass spectrometry, which allowed us to identify a unique peptide from the α chain and six unique peptides from the β chains. α-AI exhibited an optimum temperature of around 40 °C and two pH optima at 5 and 6.5, respectively. Its remarkable stability at high temperatures was measured (approximately 25% of activity retained even after 5 h at 100 °C), whereas the raw extract lost its activity entirely after just 10 min at 90 °C. Thus, the purification process significantly enhances the thermal stability of α-AI. The demonstrated effectiveness of the purified α-AI against the α-amylase enzyme in pigs, humans and insects underscores the protein's potential for treating obesity and diabetes, as well as for managing insect pests.

Enzyme-based sensor for the real-time detection of atrazine: Evidence from electrochemical and docking studies

This study focused on strategically employing the carboxylesterase enzyme Ha006a, derived from the pesticide-resistant microorganism Helicoverpa armigera, to detect atrazine. A comprehensive analysis through biochemical, biophysical and bioinformatics approaches was conducted to determine the interaction between the Ha006a protein and the herbicide atrazine. These experimental findings elucidated the potential of leveraging the inherent pesticide sequestration mechanism of the Ha006a enzyme for sensor fabrication. Numerous optimizations were undertaken to ensure the precision, reproducibility and convenient storage of the resulting electrochemical sensor, Ha006a/MCPE. This biosensor exhibited exceptional performance in detecting atrazine, demonstrating outstanding selectivity with a lower limit of detection of 5.4 µM. The developed biosensor has emerged as a reliable and cost-effective green tool for the detection of atrazine from diverse environmental samples. The Ha006a-based biosensor fabrication has expanded the possibilities for the efficient integration of insect enzymes as analytical tools, paving the way for the design of cost-effective biosensors capable of detecting and quantifying pesticides.

The Role of TcCYP6K1 and TcCYP9F2 Influences Trehalose Metabolism under High-CO2 Stress in Tribolium castaneum (Coleoptera).

Cytochrome P450 monooxygenases (CYP), crucial detoxification enzymes in insects, are involved in the metabolism of endogenous substances as well as the activation and degradation of exogenous compounds. In this study, T. castaneum was utilized to investigate the roles of TcCYP6K1 and TcCYP9F2 genes influencing in the trehalose metabolism pathway under high-CO2 stress. By predicting the functional sequences of TcCYP6K1 and TcCYP9F2 genes and analyzing their spatiotemporal expression patterns, it was discovered that both genes belong to the CYP3 group and exhibit high expression levels during the larval stage, decreasing during the pupal stage, while showing high expression in the fatty body, intestine, and malpighian tubules. Furthermore, following the knockdown of TcCYP6K1 and TcCYP9F2 genes in combination with treating larvae with 75% CO2, it was observed that larval mortality increased, and glycogen content significantly decreased, while trehalose content increased significantly. Additionally, membrane-bound trehalase enzyme activity declined, TPS gene expression was significantly upregulated, GS gene expression was significantly downregulated, and ATP content showed a marked decrease. In conclusion, CYP genes are critical responsive genes of T. castaneum to high CO2 levels, potentially impacting the insect's resistance to carbon dioxide through their involvement in the synthesis or breakdown of the carbohydrate metabolism pathway. These findings could serve as a theoretical basis for the utilization of novel pesticides in low-oxygen grain storage techniques and offer new insights for environmentally friendly pest control strategies in grain storage.

Functional Analysis of Insecticide Inhibition and Metabolism of Six Glutathione S-Transferases in the Rice Stem Borer, Chilo suppressalis.

The glutathione S-transferases (GSTs) are important detoxifying enzymes in insects. Our previous studies found that the susceptibility of Chilo suppressalis to abamectin was significantly increased when the CsGST activity was inhibited by glutathione (GSH) depletory. In this study, the potential detoxification mechanisms of CsGSTs to abamectin were explored. Six CsGSTs of C. suppressalis were expressed in vitro. Enzymatic kinetic parameters including Km and Vmax of recombinant CsGSTs were determined, and results showed that all of the six CsGSTs were catalytically active and displaying glutathione transferase activity. Insecticide inhibitions revealed that a low concentration of abamectin could effectively inhibit the activities of CsGSTs including CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1. However, the in vitro metabolism assay found that the six CsGSTs could not metabolize abamectin directly. Additionally, the glutathione transferase activity of CsGSTs in C. suppressalis was significantly increased post-treatment with abamectin. Comprehensive analysis of the results in present and our previous studies demonstrated that CsGSTs play an important role in detoxification of abamectin by catalyzing the conjugation of GSH to abamectin in C. suppressalis, and the high binding affinities of CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1 with abamectin might also suggest the involvement of CsGSTs in detoxification of abamectin via the noncatalytic passive binding and sequestration instead of direct metabolism. These studies are helpful to better understand the detoxification mechanisms of GSTs in insects.

Variation in Pesticide Toxicity in the Western Honey Bee (Apis mellifera) Associated with Consuming Phytochemically Different Monofloral Honeys

Insecticide toxicity to insect herbivores has long been known to vary across different host plants; this phenomenon has been widely documented in both foliage-feeders and sap-feeders. Species-specific phytochemical content of hostplant tissues is assumed to determine the pattern of induction of insect enzymes that detoxify insecticides, but specific phytochemicals have rarely been linked to host plant-associated variation in pesticide toxicity. Moreover, no studies to date have examined the effects of nectar source identity and phytochemical composition on the toxicity of insecticides to pollinators. In this study, we compared LD50 values for the insecticide bifenthrin, a frequent contaminant of nectar and pollen in agroecosystems, in the western honey bee, Apis mellifera, consuming three phytochemically different monofloral honeys: Nyssa ogeche (tupelo), Robinia pseudoacacia (black locust), and Fagopyrum esculentum (buckwheat). We found that bifenthrin toxicity (LD50) values for honey bees across different honey diets is linked to their species-specific phytochemical content. The profiles of phenolic acids and flavonoids of buckwheat and locust honeys are richer than is the profile of tupelo honey, with buckwheat honey containing the highest total content of phytochemicals and associated with the highest bifenthrin LD50 in honey bees. The vector fitting in the ordination analysis revealed positive correlations between LD50 values and two honey phytochemical richness estimates, Chao1 and Abundance-based Coverage Estimator (ACE). These findings suggest unequal effects among different phytochemicals, consistent with the interpretation that certain compounds, including ones that are rare, may have a more pronounced effect in mitigating pesticide toxicity.

Toxicity, biochemical and molecular docking studies of Acacia nilotica L., essential oils against insect pests

Botanical essential oils are natural insecticides derived from plants, offering eco-friendly alternatives to synthetic chemicals for pest control. In this study, the essential oils were extracted from Acacia nilotica seed cotyledons, and their toxicity was tested against insect pests. Furthermore, the chemical components of the essential oils were identified through gas chromatography-mass spectrometry (GC-MS) analysis. The essential oil extracted from A. nilotica seeds exhibited the highest mortality rates of 60% and 98% in Culex quinquefasciatus, and 60% and 96.66% mortality in Plutella xylostella at 24 and 48 h after treatment, respectively. The essential oils resulted in a lower LC50 of 159.263 ppm/mL, and LC90 of 320.930 ppm/mL within 24 h. In 48 h, the LC50 was 52.070 ppm/mL and the LC90 was 195.123 ppm/mL for C. quinquefasciatus. In the essential oil treatment of P. xylostella, the lower LC50 was 165.900 ppm/mL, and the LC90 was 343.840 ppm/mL 24 h after the treatment. At 48 h post-treatment, the LC50 decreased to 62.965 ppm/mL, and the LC90 decreased to 236.795 ppm/mL in P. xylostella. The study investigated the impact of essential oils on insect enzymes 24 h after treatment. The study revealed significant changes in the levels of insect enzymes, including a decrease in acetylcholinesterase enzymes and an increase in glutathione S-transferase compared to the control group. Essential oils had minimal effects, resulting in mortality rates of 30.66% and 46% at 24 and 48 h after treatment on Artemia salina. After 48 h, minimal toxic effects of essential oils were observed on E. eugeniae, with a mortality rate of 11.33%. The GC-MS analysis of A. nilotica seed-derived essential oils revealed ten major chemical constituents, including 6-hydroxymellein, phthalic acid, trichloroacetic acid, hexadecane, acetamide, heptacosane, eicosane, pentadecane, 1,3,4-eugenol, and chrodrimanin B. Among these constituents, Heptacosane is the major chemical component, and this molecule has a high potential for involvement in insecticidal activity.

Pathogenicity of Metarhizium rileyi (Hypocreales: Clavicipitaceae) against Tenebrio molitor (Coleoptera: Tenebrionidae).

Tenebrio molitor L., also known as the mealworm, is a polyphagous insect pest that infests various stored grains worldwide. Both the adult and larval stages can cause significant damage to stored grains. The present study focused on isolating entomopathogenic fungi from an infected larval cadaver under environmental conditions. Fungal pathogenicity was tested on T. molitor larvae and pupae for 12 days. Entomopathogenic fungi were identified using biotechnological methods based on their morphology and the sequence of their nuclear ribosomal internal transcribed spacer (ITS). The results of the insecticidal activity indicate that the virulence of fungi varies between the larval and pupal stages. In comparison to the larval stage, the pupal stage is highly susceptible to Metarhizium rileyi, exhibiting 100% mortality rates after 12 days (lethal concentration 50[LC50] = 7.8 × 106 and lethal concentration 90(LC90) = 2.1 × 1013 conidia/mL), whereas larvae showed 92% mortality rates at 12 days posttreatment (LC50 = 1.0 × 106 and LC90 = 3.0 × 109 conidia/mL). The enzymatic analyses revealed a significant increase in the levels of the insect enzymes superoxide dismutase (4.76-10.5 mg-1) and glutathione S-transferase (0.46-6.53 mg-1) 3 days after exposure to M. rileyi conidia (1.5 × 105 conidia/mL) compared to the control group. The findings clearly show that M. rileyi is an environmentally friendly and effective microbial agent for controlling the larvae and pupae of T. molitor.

Zymography of proteases in honey bees (Apis Mellifera) infected with Nosema ceranae

Nosemosis is one of the most important honey bee diseases and is caused by two fungal species of the genus Nosema, i.e., Nosema apis and Nosema ceranae. Evidence suggests that in the coming years, Nosema ceranae, which is less sensitive to fumagillin, could replace Nosema apis as the predominant pathogenic Nosema. Identifying and developing drugs that target these microsporidia without having serious effects on host bee physiology is essential for controlling nosemosis, and this requires information about bee enzymes. Serine proteases, the major protease enzymes in insects, play an important role in the insect immune system. In the present study, some proteases of honey bees infected with N. ceranae were studied by zymography. PCR tests showed that all honey bee samples were infected with N. ceranae. Also, the protein pattern of infected honey bee tissue extracts was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bands with molecular weights of 25, 35, 48, 63, 75, and 100 kDa were observed. Subsequently, the serine proteases in the bees? digestive tracts ranged in molecular weight between 23-35 kDa, and the molecular weight of cathepsins was 25 kDa by gelatin zymography. Investigation of bee proteases might be useful for acquiring more knowledge about the interaction between host and parasite and to access an effective and low-risk compound for nosemosis treatment.

Effects of ultraviolet light stress on protective and detoxification enzymes in insects

Effects of ultraviolet light stress on protective and detoxification enzymes in insects

Comparative metaproteomics reveal co-contribution of onion maggot and its gut microbiota to phoxim resistance

Pesticide resistance inflicts significant economic losses on a global scale each year. To address this pressing issue, substantial efforts have been dedicated to unraveling the resistance mechanisms, particularly the newly discovered microbiota-derived pesticide resistance in recent decades. Previous research has predominantly focused on investigating microbiota-derived pesticide resistance from the perspective of the pest host, associated microbes, and their interactions. However, a gap remains in the quantification of the contribution by the pest host and associated microbes to this resistance. In this study, we investigated the toxicity of phoxim by examining one resistant and one sensitive Delia antiqua strain. We also explored the critical role of associated microbiota and host in conferring phoxim resistance. In addition, we used metaproteomics to compare the proteomic profile of the two D. antiqua strains. Lastly, we investigated the activity of detoxification enzymes in D. antiqua larvae and phoxim-degrading gut microbes, and assessed their respective contributions to phoxim resistance in D. antiqua. The results revealed contributions by D. antiqua and its gut bacteria to phoxim resistance. Metaproteomics showed that the two D. antiqua strains expressed different protein profiles. Detoxifying enzymes including Glutathione S-transferases, carboxylesterases, Superoxide Dismutase, Glutathione Peroxidase, and esterase B1 were overexpressed in the resistant strain and dominated in differentially expressed insect proteins. In addition, organophosphorus hydrolases combined with a group of ABC type transporters were overexpressed in the gut microbiota of resistant D. antiqua compared to the sensitive strain. 85.2% variation of the larval mortality resulting from phoxim treatment could be attributed to the combined effects of proteins from both from gut bacteria and D. antiqua, while the individual contribution of proteins from gut bacteria or D. antiqua alone accounted for less than 10% of the variation in larval mortality caused by phoxim. The activity of the overexpressed insect enzymes and the phoxim-degrading activity of gut bacteria in resistant D. antiqua larvae were further confirmed. This work enhances our understanding of microbiota-derived pesticide resistance and illuminates new strategies for controlling pesticide resistance in the context of insect-microbe mutualism.

Characterization of the TcCYPE2 Gene and Its Role in Regulating Trehalose Metabolism in Response to High CO2 Stress

Cytochrome P450 monooxygenase (CYP) is one of the three detoxification metabolic enzymes in insects, and is involved in the metabolism and transformation of endogenous substances as well as the activation and degradation of exogenous compounds. This study aims to reveal the molecular mechanism of CYP9E2 in Tribolium castaneum in adapting to high-CO2 stress. By predicting the sequence function of CYP9E2, analyzing the temporal and spatial expression profile of TcCYP9E2, and using RNAi to silence TcCYP9E2 combined with a high-CO2 stress treatment, we measured the carbohydrate content, trehalase activity, and gene expression levels in trehalose metabolism of T. castaneum. A bioinformatics analysis showed that the predicted molecular weight of the protein encoded by TcCYP9E2 is 60.15, the theoretical isoelectric point is 8.63, there is no signal peptide, and the protein is hydrophilic. An evolutionary tree analysis showed that TcCYP9E2 belongs to the CYP6 family and belongs to the CYP3 group; and the spatiotemporal expression profile results showed that TcCYP9E2 was highly expressed in the larvae midgut 48 h after injection of dsCYP9E2, with survival rates decreasing with the increase in CO2 concentration. Under the condition of 75% CO2, the contents of glycogen, glucose, ATP, and membrane-bound trehalase decreased significantly after the injection of dsCYP9E2. The expression of TRE-1, TRE-2, and GP in trehalose metabolism and energy pathways was significantly downregulated.

The role of P450 enzymes in malaria and other vector-borne infectious diseases.

Vector-borne infectious diseases are still an important global health problem. Malaria is the most important among them, mainly pediatric, life-threatening disease. Malaria and other vector-borne disorders caused by parasites, bacteria, and viruses have a strong impact on public health and significant economic costs. Most vector-borne diseases could be prevented by vector control, with attention to the ecological and biodiversity conservation aspects. Chemical control with pesticides and insecticides is widely used as a measure of prevention although increasing resistance to insecticides is a serious issue in vector control. Metabolic resistance is the most common mechanism and poses a big challenge. Insect enzyme systems, including monooxygenase CYP P450 enzymes, are employed by vectors mainly to metabolize insecticides thus causing resistance. The discovery and application of natural specific inhibitors/blockers of vector P450 enzymes as synergists for commonly used pesticides will contribute to the "greening" of insecticides. Besides vector CYPs, host CYP enzymes could also be exploited to fight against vector-borne diseases: using mostly their detoxifying properties and involvement in the immune response. Here, we review published research data on P450 enzymes from all players in vector-borne infections, that is, pathogens, vectors, and hosts, regarding the potential role of CYPs in disease. We discuss strategies on how to exploit cytochromes P450 in vector-borne disease control.

Effectiveness of ethyl acetate extract from Aspergillus flavipes AUMC 11390 culture filtrate on biological and physiological performance of the spiny bollworm, Earias insulana, (Boisd.) (Lepidoptera: Nolidae)

BackgroundSpiny bollworm, Earias insulana is a serious cotton pest in Egypt. Besides the economic losses it caused, treatment with chemical insecticides has negative effects on human health and the environment, thus the development of a powerful safe control strategy rather than chemical pesticides is an international goal.ResultsEthyl acetate extract from Aspergillus flavipes AUMC 11390 culture filtrate has an insecticidal activity against E. insulana causing larval and pupal mortality of 58.33, and 15.59%, respectively, compared with controls, in addition, reduction in adult's emergency and deformation of emerged adults. The impact of fungal extract treatment extended to adult stages by diminishing the male and the female longevity, the number of produced eggs and the hatchability percent. Furthermore, A. flavipes AUMC 11390 ethyl acetate extract caused a strong disturbance on some insect enzymes including amylase, invertase, trehalase, GOT, GPT and acetylcholinesterase, alongside total lipid and total protein. Analysis of ethyl acetate fungal extract revealed the presence of one hydrocarbon 3-Eicosene and four long-chain alcohols namely hexadecanol, 1-hexadecanol, 1-octadecanol, and 1-pentadecanol which are known for their insecticidal activity.ConclusionA. flavipes AUMC 11390 culture filtrate might represent a promising source for different important bioactive compounds that could be used as a potential biocontrol agent involved in E. insulana management strategies.

In Vitro and Molecular Docking Evaluation of Larvicidal Effects of Essential Oils of Five Aromatic Plants on the Fall Armyworm Spodoptera frugiperda JE. Smith (Lepidoptera: Noctuidae) from Ivory Coast.

Faced with the serious consequences resulting from the abusive and repeated use of synthetic chemicals, today rethinking crop protection is more than necessary. It is in this context that the essential oils of the Lamiaceae Ocimum gratissimum and Ocimum canum, the Poaceae Cymbopogon citratus and nardus and a Rutaceae Citrus sp. of known chemical compositions were experimented. The evaluation of the larvicidal potential of the essential oils was done by the method of topical application of the test solutions, on the L1-L2 stage larvae from the first generation of S. frugiperda obtained after rearing in an air-conditioned room. Lethal concentrations (LC10 , LC50 and LC90 ) were determined after 48 h. After assessing the larvicidal potential of essential oils, molecular docking was carried out to study protein-ligand interactions and their propensity to bind to insect enzyme sites (AChE). The essential oil of O. gratissimum was the most effective with the lowest lethal concentrations (LC10 =0.91 %, LC50 =1.91 % and LC90 =3.92 %). The least toxic oil to larvae was Citrus sp. (LC10 =5.44 %, LC50 =20.50 % and LC90 =77.41 %). Molecular docking revealed that p-cymene and thymol from O. gratissimum essential oil are structurally similar and bind to the AChE active site via predominantly hydrophobic interactions and a H-bond with Tyr374 in the case of thymol. The essential oil of O. gratissimum constitutes a potential candidate for the development of biological insecticides for the fight against insect pests and for the protection of the environment.

Aliens in the CYPome of the black fungus gnat, Bradysia coprophila

The diverse cytochrome P450 enzymes of insects play essential physiological roles and also play important roles in the metabolism of environmental chemicals such as insecticides. We manually curated the complement of P450 (CYP) genes, or CYPome, of the black fungus gnat, Bradysia (Sciara) coprophila (Diptera, Sciaroidea), a species with a variable number of chromosomes. This CYPome carries two types of “alien” P450 genes. The first type of alien P450s was found among the 163 CYP genes of the core genome (autosomes and X). They consist of 28 sequences resulting from horizontal gene transfer, with closest sequences not found in insects, but in other arthropods, often Collembola. These genes are not contaminants, because they are expressed genes with introns, found in synteny with regular dipteran genes, also found in B. odoriphaga and B. hygida. Two such “alien” genes are representatives of CYP clans not otherwise found in insects, a CYP53 sequence related to fungal CYP53 genes, and a CYP19-like sequence similar to some collembolan sequences but of unclear origin. The second type of alien P450s are represented by 99 sequences from germline-restricted chromosomes (GRC). While most are P450 pseudogenes, 33 are apparently intact, with half being more closely related to P450s from Cecidomyiidae than from Sciaridae, thus supporting the hypothesis of a cross-family hybridization origin of the GRC.

Glutathione S-Transferase Highly Expressed in Holotrichia parallela Antennae Inactivates the Odorant Unsaturated Aldehyde Volatiles.

Odorant-degrading enzymes in insects play a vital role in maintaining olfactory sensitivity. However, the role and molecular mechanism of glutathione S-transferases (GSTs) in odorant inactivation has been rarely studied. In the present study, 31 GSTs were identified from the antennal transcriptome of Holotrichia parallela. HpGSTd1 possesses the highest transcriptome expression level. Recombinant HpGSTd1 showed degradation activity toward various unsaturated aldehyde volatiles. Furthermore, the metabolite of cinnamaldehyde was identified by high-resolution mass spectrometry (HRMS). The molecular docking analysis and site-directed mutagenesis revealed the key residues of HpGSTd1 in degrading odorants. In addition, the unsaturated aldehyde volatiles elicited the behavioral and electrophysiological responses of H. parallela. Taken together, our findings suggest that HpGSTd1 may play an essential role in inactivating odorants in H. parallela, which provides new insights for identifying molecular targets and exploring effective olfactory regulators for this underground pest.

ИЗУЧЕНИЕ МЕХАНИЗМОВ ДЕТОКСИКАЦИИ ПРОИНСЕКТИЦИДОВ У РЫЖЕГО ТАРАКАНА BLATTELLA GERMANICA

The data on the observed detoxification process of the proinsecticides indoxacarb and chlorfenapyr in the body of the German cockroach, Blattella germanica of several laboratory strains are presented. Different resistance ratios to indoxacarb of M10 (16.7×), DM (45.8×) and MAG (10.4×) strains, and chlorfenapyr (6.7×, 2.0×, 1.7×, respectively) against the background of high resistance to cypermethrin (92–833×), fipronil (46–192×), tolerance to chlorpyrifos (5.0–50×) and thiamethoxam (3.5–10×). The insecticidal activity of indoxacarb and chlorfenapyr was studied with desynchronized application of synergists PPB, TBTP, DEM - inhibitors of insect enzyme systems and an inhibitor of ABC transporters verapamil. The antagonistic action of synergists indicates the difficulty of converting proinsecticides into a toxic metabolite. In the M10 strain, PPB and TBTP significantly suppressed the activity of chlorfenapyr, while in DM and MAG strains, activity of chlorfenapyr suppressed only under the action of TBTP. For the DM strain most resistant to indoxacarb, the use of TBTP and DEM reduces resistance by 2.8 and 2.4 times, respectively. Verapamil has a statistically significant effect on susceptibility to chlorfenapyr only in S-NIID and DM strains, and to indoxacarb in MAG and DM strains. Key words: indoxacarb, chlorfenapyr, German cockroach, resistanc.

First report on the enzymatic and immune response of Metarhizium majus bag formulated conidia against Spodoptera frugiperda: An ecofriendly microbial insecticide.

Entomopathogenic fungi from microbial sources are a powerful tool for combating insecticide resistance in insect pests. The purpose of the current study was to isolate, identify, and evaluate bag-formulated entomopathogenic fungal conidial virulence against insect pests. We further investigated the enzymatic responses induced by the entomopathogenic fungi as well as the effect on a non-target species. Entomopathogenic fungi were isolated from the Palamalai Hills, India, using the insect bait method, and the Metarhizium majus (MK418990.1) entomopathogen was identified using biotechnological techniques (genomic DNA isolation and 18S rDNA amplification). Bag-formulated fungal conidial efficacy (2.5 × 103, 2.5 × 104, 2.5 × 105, 2.5 × 106, and 2.5 × 107 conidia/ml) was evaluated against third instar larvae of Spodoptera frugiperda at 3, 6, 9, and 12 days of treatment, and acid and alkaline phosphatases, catalase, and superoxide dismutase enzymatic responses were evaluated at 3 days post-treatment. After 12 days of treatment, non-target assays on the earthworm Eudrilus eugeniae were performed using an artificial soil assay. Results of the bag formulated fungal conidial treatment showed that S. frugiperda had high susceptibility rates at higher concentrations (2.5 × 107 conidia/ml) of M. majus. Lower concentration of 2.5 × 103 conidia/ml caused 68.6% mortality, while 2.5 × 107 conidia/ml caused 100% mortality at 9 days post treatment. Investigation into enzymatic responses revealed that at 3 days post M. majus conidia exposure (2.5 × 103 conidia/ml), insect enzyme levels had significantly changed, with acid and alkaline phosphatases, and catalase enzymes significantly reduced and superoxide dismutase enzymes significantly raised relative to the control. After 12 days of treatment, no sublethal effects of M. majus conidia were observed on E. eugeniae, with no observed damage to gut tissues including lumen and epithelial cells, the nucleus, setae, coelom, mitochondria, and muscles. This study offers support for the use of fungal conidia in the target-specific control of insect pests.

Impact of Insecticides at Sublethal Concentrations on the Enzyme Activities in Adult Musca domestica L.

Nowadays, the use of pesticides is, as before, the most common way to control arthropod plant pests and the ectoparasites of animals. The sublethal effects of pesticides on insects can appear at different levels, from genetics to populations, and the study of these effects is important for a better understanding of the environmental and evolutionary patterns of pesticidal resistance. The current study aimed to assess the sublethal effects of chlorfenapyr and fipronil on the activities of detoxifying enzymes (carboxylesterase-CarE, acetylcholinesterase-AChE, glutathione-S-transferase-GST, and cytochrome P450 monooxygenase-P450) in adults Musca domestica L. The insects were exposure to insecticides by a no-choice feeding test and the enzyme activities and the AChE kinetic parameters were examined in female and male specimens at 24 h after their exposure. According to Tukey's test, the CarE activity was statistically significantly decreased by 29.63% in the females of M. domestica after an exposure to chlorfenapyr at a concentration of 0.015% when compared to the controls (p ≤ 0.05). An exposure to the sublethal concentration of fipronil (0.001%) was followed by a slightly decrease in the specific activity (33.20%, p ≤ 0.05) and the main kinetic parameters (Vmax, Km) of AChE in females in comparison with the control values. The GST and P450 activities had not significantly changed in M. domestica males and females 24 h after their exposure to chlorfenapyr and fipronil at sublethal concentrations. The results suggest that the males and females of M. domestica displayed biochemically different responses to fipronil, that is a neurotoxin, and chlorfenapyr, that is a decoupler of oxidative phosphorylation. Further research needs to be addressed to the molecular mechanisms underlying the peculiarities of the insect enzyme responses to different insecticides.