Molecular and functional characterization of heat-shock protein 70 in Aphis gossypii under thermal and xenobiotic stresses.

Aphis gossypii, a globally distributed and economically significant pest of several crops, is known to infest a wide range of host plants. Heat shock proteins (Hsps), acting as molecular chaperones, are essential for the insect's environmental stress responses. The present study investigated the molecular characteristics and expression patterns of AgHsp70, a heat shock protein gene, in Aphis gossypii. Our phylogenetic analysis revealed that AgHsp70 shared high similarity with homologs from other insects, suggesting a conserved function across species. The developmental expression profiles of AgHsp70 in A. gossypii showed that the highest transcript levels were observed in the fourth instar nymphs, while the lowest levels were detected in the third instar nymphs. Heat stress and exposure to four different xenobiotics (2-tridecanone, tannic acid, gossypol, and flupyradifurone (4-[(2,2-difluoroethyl)amino]-2(5H)-furanone)) significantly up-regulated AgHsp70 expression. Knockdown of AgHsp70 using RNAi obviously increased the susceptibility of cotton aphids to 2-tridecanone, gossypol and flupyradifurone. Dual-luciferase reporter assays revealed that gossypol and flupyradifurone significantly enhanced the promoter activity of AgHsp70 at a concentration of 10 mg/L. Furthermore, we identified the transcription factor heat shock factor (HSF) as a regulator of AgHsp70, as silencing AgHSF reduced AgHsp70 expression. Our results shed light on the role of AgHsp70 in xenobiotic adaptation and thermo-tolerance.

The gut symbiont Sphingomonas mediates imidacloprid resistance in the important agricultural insect pest Aphis gossypii Glover.

BACKGROUND: Neonicotinoid insecticides are applied worldwide for the control of agricultural insect pests. The evolution of neonicotinoid resistance has led to the failure of pest control in the field. The enhanced detoxifying enzyme activity and target mutations play important roles in the resistance of insects to neonicotinoid resistance. Emerging evidence indicates a central role of the gut symbiont in insect pest resistance to pesticides. Existing reports suggest that symbiotic microorganisms could mediate pesticide resistance by degrading pesticides in insect pests. RESULTS: The 16S rDNA sequencing results showed that the richness and diversity of the gut community between the imidacloprid-resistant (IMI-R) and imidacloprid-susceptible (IMI-S) strains of the cotton aphid Aphis gossypii showed no significant difference, while the abundance of the gut symbiont Sphingomonas was significantly higher in the IMI-R strain. Antibiotic treatment deprived Sphingomonas of the gut, followed by an increase in susceptibility to imidacloprid in the IMI-R strain. The susceptibility of the IMI-S strain to imidacloprid was significantly decreased as expected after supplementation with Sphingomonas. In addition, the imidacloprid susceptibility in nine field populations, which were all infected with Sphingomonas, increased to different degrees after treatment with antibiotics. Then, we demonstrated that Sphingomonas isolated from the gut of the IMI-R strain could subsist only with imidacloprid as a carbon source. The metabolic efficiency of imidacloprid by Sphingomonas reached 56% by HPLC detection. This further proved that Sphingomonas could mediate A. gossypii resistance to imidacloprid by hydroxylation and nitroreduction. CONCLUSIONS: Our findings suggest that the gut symbiont Sphingomonas, with detoxification properties, could offer an opportunity for insect pests to metabolize imidacloprid. These findings enriched our knowledge of mechanisms of insecticide resistance and provided new symbiont-based strategies for control of insecticide-resistant insect pests with high Sphingomonas abundance.

The sulfoximine insecticide sulfoxaflor exposure reduces the survival status and disrupts the intestinal metabolism of the honeybee Apis mellifera.

Honeybees (Apis mellifera) are indispensable pollinators in agricultural production, biodiversity conservation, and nutrients provision. The abundance and diversity of honeybees have been rapidly diminishing, possibly related to the extensive use of insecticides in ecosystems. Sulfoxaflor is a novel sulfoximine insecticide that, like neonicotinoids, acts as a competitive modulator of nicotinic acetylcholine receptors (nAChR) in insects. However, few studies have addressed the negative effects of sulfoxaflor on honeybees at environmentally relevant concentrations. In the present study, adult workers were fed a 50% (w/v) of sugar solution containing different concentrations (0, 0.05, 0.5 and 2.0 mg/L) of sulfoxaflor for two weeks consecutively. The survival rates, food intake, and body weight of the honeybees significantly decreased after continuous exposure at higher doses (0.5 and 2.0 mg/L) of sulfoxaflor when compared with the control. The change in the metabolites in the honeybee gut was determined using high-throughput non-targeted metabolomics on day 14 after sulfoxaflor treatment. The results revealed that 24 and 105 metabolites changed after exposure to 0.5 and 2.0 mg/L sulfoxaflor, respectively, compared with that of the control groups. A total of 12 changed compounds including pregenolone and glutathione were detected as potential biomarkers, which were eventually found to be enriched in pathways of the steroid hormone biosynthesis (p = 0.0001) and glutathione metabolism (p = 0.021). These findings provide a new perspective on the physiological influence of sulfoxaflor stress in honeybees.

Establishment of Toxicity and Susceptibility Baseline of Broflanilide for Aphis gossypii Glove.

The Aphis gossypii is an important pest that can damage cotton plants and can cause a huge economic loss worldwide. Chemical control is a main method to manage this pest, but the cotton aphid resistance to insecticides has become a severe problem in the management of the cotton aphid. It is important to introduce a novel insecticide for rotational application with other insecticides. Broflanilide, as a meta-diamide insecticide with a special mode of action, showed high efficiency against lepidopterous larvae. However, we found that broflanilide possessed high insecticidal activity against the sap-sucking pest A. gossypii. The susceptibility of A. gossypii to broflanilide from 20 field populations in main cotton planting areas of China in 2021 was determined by the leaf-dipping method. LC50 values of broflanilide to A. gossypii ranged from 0.20 µg mL-1 to 1.48 µg mL-1. The susceptible baseline of A. gossypii to broflanilide was established with the LC50 value of 0.41 µg mL-1 and might be used to calculate the resistance ratio (RR) of cotton aphid population in broflanilide resistance monitoring. The RR value of field populations in China was from 0.49 to 3.61 in 2021. It suggested that the broflanilide may be a potential agent in the resistance management of A. gossypii to insecticides. These results are significantly useful for the rational chemical control of cotton aphids.

Status of the Resistance of Aphis gossypii Glover, 1877 (Hemiptera: Aphididae) to Afidopyropen Originating from Microbial Secondary Metabolites in China.

The resistance of cotton aphids to various forms of commonly used pesticides has seriously threatened the safety of the cotton production. Afidopyropen is a derivative of microbial metabolites with pyropene insecticide, which has been shown to be effective in the management of Aphis gossypii. Several field populations of Aphis gossypii were collected from the major cotton-producing regions of China from 2019 to 2021. The resistance of these populations to afidopyropen was estimated using the leaf-dipping method. The LC50 values of these field populations ranged from 0.005 to 0.591 mg a.i. L-1 in 2019, from 0.174 to 4.963 mg a.i. L-1 in 2020 and from 0.517 to 14.16 mg a.i. L-1 in 2021. The resistance ratios for all A. gossypii populations ranged from 0.03 to 3.97 in 2019, from 1.17 to 33.3 in 2020 and from 3.47 to 95.06 in 2021. The afidopyropen resistance exhibited an increasing trend in the field populations of Cangzhou, Binzhou, Yuncheng, Kuerle, Kuitun, Changji and Shawan from 2019 to 2021. This suggests that the resistance development of the cotton aphid to afidopyropen is inevitable. Therefore, it is necessary to rotate or mix afidopyropen with other insecticides in order to inhibit the development of afidopyropen resistance in field populations.

Toxicity and risk assessment of nine pesticides on nontarget natural predator Harmonia axyridis (Coleoptera: Coccinellidae).

BACKGROUND: Harmonia axyridis (Coleoptera: Coccinellidae) is a beneficial predatory arthropod in the agricultural ecosystem. For the success and development of integrated pest management strategies, it is essential to assess the toxicity risks of commonly used pesticides to nontarget arthropods. RESULTS: The glass tube residue method was used to determine the risk of nine pesticides to H. axyridis after second-instar exposure. To assess the potential risk of the selected pesticides, the pre-adult LR50 values were calculated by the hazard quotient (HQ) method. The LR50 (application rate causing 50% mortality) values of imidacloprid, dinotefuran, thiamethoxam, acetamiprid, bifenthrin, and dimethoate were 0.44, 0.82, 0.10, 0.05, 0.04, and 0.21 g a.i. ha-1 , respectively, showing unacceptable risk to H. axyridis after exposure to in and off field. However, emamectin benzoate and two fungicides, tebuconazole and myclobutanil, posed a low risk to H. axyridis and their HQ values were less than the trigger value of 5. CONCLUSIONS: The four neonicotinoid insecticides (imidacloprid, dinotefuran, thiamethoxam, and acetamiprid), pyrethroid bifenthrin, and organophosphorus dimethoate showed a high risk to H. axyridis. Emamectin benzoate, tebuconazole, and myclobutanil showed a low risk to H. axyridis under both exposure scenarios. The results provide critical scientific evidence to guide future regulation of pesticide management practices and protection of nontarget arthropods like H. axyridis. © 2022 Society of Chemical Industry.

Comparative sensitivity of Neoseiulus cucumeris and its prey Tetranychus cinnabarinus, after exposed to nineteen pesticides.

The toxicity tests of nineteen commonly used pesticides were carried out to compare the sensitivity differences between predatory mite Neoseiulus cucumeris and its prey Tetranychus cinnabarinus by a "leaf spray method" in laboratory microcosms. For two avermectins, emamectin benzoate and abamectin, exhibited high bioactivity against T. cinnabarinusf with LR50 values of 0.04 and 0.05 g a.i./ha, respectively, but these two insecticides showed the opposite toxic effects to N. cucumeris. These two agents showed strong selectivity for the two test species with Selective Toxicity Rate (STR) values of 950 and 620, respectively. However, for five neonicotinoids, the LR50s of dinotefuran, thiamethoxam, imidacloprid, and acetamiprid were all greater than the recommended rates in the field except for clothianidin, and they showed no obvious toxicity difference to the two species with STR values ranging from 0.58 to 2.00. For two organophosphates, malathion is more toxic to N. cucumeris than T. cinnabarinus, however, dimethoate showed a higher toxic effect on T. cinnabarinus. In addition, the toxicity of four pyrethroids, bifenthrin, deltamethrin, cyhalothrin and gamma-cyhalothrin to N. cucumeris was higher than that of T. cinnabarinus, except for alpha-cypermethrin. For five acaricides, spirodiclofen, spirotetramat and pyridaben had no obvious selectivity to the two organisms, while diafenthiuron and chlorfenapyr were found to be highly toxic to T. cinnabarinus than N. cucumeris with STR values of 14.2 and 68.5, respectively. Thus, some pesticides above-mentioned like emamectin benzoate, abamectin, diafenthiuron and chlorfenapyr exhibited potential to be used in the management programs of T. cinnabarinus, especially in organically based production systems where there are fewer chemical control measures available, which need to combine with natural enemies to achieve the best control effect.

Sublethal and transgenerational effects of acetamiprid and imidacloprid on the predatory bug Orius sauteri (Poppius) (Hemiptera: Anthocoridae).

The predatory bug Orius sauteri (Poppius) is currently one of the most important beneficial arthropods in Northeast Asia and used as a biological control agent of several small pest arthropods including Frankliniella occidentalis (Pergande). Two neonicotinoid chemical insecticides, acetamiprid and imidacloprid, mainly used in China as chemical control on F. occidentalis, although applied at sublethal concentrations in the field or greenhouse to protect beneficial arthropods, still may affect the predator O. sauteri. The objective of present work is to assess the long-term effects of 24-h exposure time to these two insecticides on the life-cycle of O. sauteri at application rates similar to the laboratory 24-h LC10, LC20 and LC30 of O. sauteri. Results showed that acetamiprid and imidacloprid at all tested concentrations significantly decreased the fecundity of O. sauteri females, while the effect of acetamiprid was higher than that of imidacloprid. Moreover, the oviposition period and longevity of O. sauteri to both insecticides shortened. The sublethal effects on the first progeny (F1 generation) were also found to increase nymphal mortality, shorten adult longevity and reduce fecundity. However, all treatments of acetamiprid and imidacloprid at the concentration of LC10, LC20 and LC30 caused no significant effect on the developmental time of different nymphal stages and sex ratio of the F1 generation. This paper is the first one that assesses the compatibility between neonicotinoid insecticides and O. sauteri, and shows that the application of acetamiprid and imidacloprid likely interferes with the population dynamic of O. sauteri.

Characteristics of biochars prepared by co-pyrolysis of sewage sludge and cotton stalk intended for use as soil amendments.

The safe disposal and utilisation of sewage sludge can be challenging because of the potential environmental risks posed by heavy metals in the sludge. Conversion of sewage sludge and agriculture biomass into biochars that can be used to improve or remediate contaminated soils is a promising solution to this problem. In this study, biochars were produced via co-pyrolysis of sewage sludge and cotton stalk (1:1, w/w) at temperatures ranging from 300°C to 600°C. Then, the potential environmental risks of heavy metals and properties of the biochars were investigated. The addition of cotton stalk promoted the migration and transformation of heavy metals from bioavailable to stable fractions, which significantly reduced the potential environmental risks of heavy metals in biochars. Moreover, compared with biochars obtained via pyrolysis of sewage sludge alone, the pH values, C contents, and adsorption capacities of biochars increased, while the yields, ash contents, specific surface areas and molar H/C ratios decreased. In summary, co-pyrolysis of sewage sludge and cotton stalk is a feasible method for alleviating the potential environmental risks of heavy metals in biochars used to treat soils.

Effects of toxic ß-glucosides on carbohydrate metabolism in cotton bollworm, Helicoverpa armigera (Hübner).

The purpose of this study was to evaluate the effects of three toxic ß-glucosides, phlorizin, santonin, and amygdalin, on carbohydrate metabolism in the cotton bollworm, Helicoverpa armigera (Hübner), when diets mixed with ß-glucosides were fed to third-instar larvae. The growth of the larvae was significantly inhibited by exposure to santonin after 96 hr but not obviously affected by phlorizin and amygdalin. The midgut trehalase activities were only 51.7%, 32%, and 42.5% of that of the control after treatment with phlorizin, santonin and amygdalin at 2 mg/ml, respectively. In the hemolymph and fat body, the amount of trehalose decreased in all cases. However, the effects of santonin on the alteration of the glycogen and glucose levels as well as the activities of glycogen phosphorylase, were different than those of the other two ß-glucosides. It appears that the three ß-glucosides have different influences on the carbohydrate metabolism of cotton bollworm.

Comparative susceptibility of thirteen selected pesticides to three different insect egg parasitoid Trichogramma species.

The parasitoid Trichogramma species are indispensable natural enemies of many lepidopterans and it plays an important role in integrated pest management (IPM) programs throughout the world. Laboratory studies were conducted to compare the susceptibility of three Trichogramma egg parasitoid species to ten common insecticides and three herbicides. The adults of Trichogramma dendrolimi, T. chilonis, and T. ostriniae were exposed to the above-mentioned pesticides by a glass-vial residue method. Among the four neonicotinoids, dinotefuran and thiamethoxam exhibited extremely toxic effects on the Trichogramma dendrolimi and T. chilonis, with Risk Quotient (RQ) values ranging from 1471.2 to 5492.5. However, these two neonicotinoids have a relatively low toxicity to T. ostriniae, with RQ values 433.6 and 915.4, respectively. In addition, Imidacloprid and acetamiprid were slightly to moderately toxic to all the tested parasitic wasps and their RQ values are less than 500. For pyrethroids, all the selected compounds were slightly to moderately toxic to three Trichogramma species except that cyhalothrin was dangerously toxic to T. dendrolimi and T. chilonis, with RQ values 2567.6 and 3950.4. Among the three herbicides tested, pendimethalin, butralin and napropamid were slightly to moderately toxic to egg parasitoids, with all RQ values below 1000. For two avermectins, abamectin were slightly to moderately toxic to all three wasps with RQ values 635.6, 148.3 and 254.2, respectively. However, emamectin benzoate was found to be safe for the parasitoids. Furthermore, T. dendrolimi showed higher sensitivity than T. chilonis and T. ostriniae to the pesticides based on the comparison of LR50 (application rate causing 50% mortality) values. The present results provide informative data for implementing biological and chemical control strategies in integrated pest management.

Effects of acetamiprid on life cycle development of predatory mite Amblyseius cucumeris (Acari: Phytoseiidae) after contact exposure.

Amblyseius cucumeris (Oudemans) is a beneficial non-target arthropod (NTA) and a key predator of pest mites in integrated pest management (IPM) programs across china. The toxic effects of insecticides have been extensively reported on predatory mites, but few studies devoted to the toxicity of compounds to A. cucumeris. In this study, the effects of a single application of acetamiprid against the A. cucumeris were investigated in a 48-h acute and a 30-d chronic test. In both tests the insecticide acetamiprid was applied once. In the acute test, the "open glass plate method" was used, with a 48-h LC50 value of 223.6 (149.8-336.9) mg a. i. L-1 for adult female. The LC50 was 1.49-fold the Maximum field recommended concentration. In the chronic test, exposure concentrations (1.12, 2.24, 4.47, 8.94, and 22.4 mg a. i. L-1) were designed based on a preliminary 48-h LC50 value for adult female. Above treatment levels of 2.24-4.47 mg a. i. L-1 reduce the total development time, survival rates and food consumption of A. cucumeris in different development stages. In addition, at acetamiprid concentrations higher than 2.24 mg a. i. L-1 serious effect on the capacity of oviposition of female adults were observed. However, even the highest treatment level of 22.4 mg a. i. L-1 did not affect egg hatching rates of exposed eggs. Acetamiprid had significant adverse effects on different development life stages of A. cucumeris. The results provide informative data for implementing biological and chemical control strategies in integrated pest management of spider mites.

Toxicity of six insecticides to predatory mite Amblyseius cucumeris (Oudemans) (Acari: Phytoseiidae) in- and off-field.

Amblyseius cucumeris (Oudemans) is a beneficial non-target arthropod (NTA) and a key predator of tetranychid mites in integrated pest management (IPM) programs across China. Evaluating the toxic effects of insecticides on such predatory mites is essential for the success and development of IPM. We tested six insecticides to determine the risk of neonicotinoid insecticide toxicity to predatory mites, using the 'open glass plate method' and adult female A. cucumeris in a "worst case laboratory exposure" scenario. A 48-h toxicity test was performed using the hazard quotient (HQ) approach to evaluate the risk of each insecticide. The LR50 values (application rate that caused 50% mortality) of acetamiprid, thiamethoxam, imidacloprid, and dinotefuran were 76.4, 104.5, 84.9, and 224.6 g active ingredient (a.i.) ha-1, respectively, with in-field HQ values of 0.40, 1.28, 0.49, and 0.82, respectively. The HQ values were lower than the trigger value of 2, and were consistent with off-field values. The risks of the four neonicotinoid insecticides to adult female A. cucumeris were acceptable in two exposure scenarios in field and off field. The 48-h LR50 values for bifenthrin and malathion were 0.008 and 0.062 g. a.i. ha-1, respectively, which were much lower than the recommended field application rates. The HQ values were much higher than the trigger values for both in- and off-field, indicating that the risks of these two insecticides were unacceptable. Bifenthrin and malathion posed an extremely high risk to the test species, and their use should be restricted to reduce risks to the field with augmentative releases of A. cucumeris.

Gene Cloning, Prokaryotic Expression, and Biochemical Characterization of a Soluble Trehalase in Helicoverpa armigera Hübner (Lepidoptera: Noctuidae).

Trehalase is an indispensable component of insect hemolymph that plays important role in energy metabolism and stress resistance. In this study, we cloned and expressed the gene encoding soluble trehalase (HaTreh-1) of Helicoverpa armigera (cotton bollworm) and characterized the enzyme. HaTreh-1 had a full-length open reading frame encoding a protein of 571 amino acids. Sequence comparison indicated that HaTreh-1 was similar to some known insect trehalases. Two essential active sites (D321 and E519) and three essential residues (R168, R221, and R286) were conserved in HaTreh-1. The recombinant trehalase was expressed in Escherichia coli and purified by nickel exchange chromatography. Molecular weight of the recombinant protein was about 71 kDa, and the optimum HaTreh-1 enzyme activity is at 55°C with pH 6.0. Enzymatic assays showed a Km value of 72.8 mmol/liter and a Vmax value of 0.608 mmol/(liter·min). Inhibition assays in vitro indicated that castanospermine, a polyhydroxylated alkaloid, was an effective competitive inhibitor of trehalase with a Ki value of 6.7 µmol/liter. The inhibitor action of castanospermine was linked to its modification effect on trehalase structure. The circular dichroism spectrum showed that the percentage of α-helix increased under the presence of castanospermine. Results of our study will aid in developing effective trehalase inhibitors for controlling H. armigera in the future.