Deciphering the impact of greenhouse pesticides on hepatic metabolism profile: Toxicity experiments on HepG2 cells using chlorpyrifos and emamectin benzoate.

Epidemiological evidence on the health effects of pesticide exposure among greenhouse workers is limited, and the mechanisms are lacking. Building upon our team's previous population study, we selected two pesticides, CPF and EB, with high detection rates, based on the theoretical foundation that the liver serves as a detoxifying organ, we constructed a toxicity model using HepG2 cells to investigate the impact of individual or combined pesticide exposure on the hepatic metabolism profile, attempting to identify targeted biomarkers. Our results showed that CPF and EB could significantly affect the survival rate of HepG2 cells and disrupt their metabolic profile. There were 117 metabolites interfered by CPF exposure, which mainly affected ABC transporter, biosynthesis of amino acids, center carbon metabolism in cancer, fatty acid biosynthesis and other pathways, 95 metabolites interfered by EB exposure, which mainly affected center carbon metabolism in cancer, HIF-1 signaling pathway, valine, leucine and isoleucine biosynthesis, fatty acid biosynthesis and other pathways. The cross analysis and further biological experiments confirmed that CPF and EB pesticide exposure may affect the HIF-1 signaling pathway and valine, leucine and isoleucine biosynthesis in HepG2 cells, providing reliable experimental evidence for the prevention and treatment of liver damage in greenhouse workers.

New insights into resveratrol attenuates hepatotoxicity in emamectin benzoate-exposed grass carp (Ctenopharyngodon idella) via NO system/NF-κB signaling pathway.

Emamectin benzoate (EMB) is extensively used as a crop protection agent. Overuse of EMB poses a serious threat to the quality of water and non-target organisms in the environment. Resveratrol (RES) is a natural phytoalexin with the function of anti-oxidation and anti-inflammation. Nonetheless, it is unclear whether EMB affects the expression of cytokines and induces autophagy, apoptosis, and necroptosis of hepatocytes (L8824 cell) in grass carp (Ctenopharyngodon idella), and whether RES has an attenuate function in this process. Therefore, we established the L8824 cells model of EMB exposure and treated it with RES. The results showed that compared with the control (CON) group, EMB exposure significantly increased the nitric oxide (NO) content, inducible nitric oxide synthase (iNOS) activity, and the expression of iNOS and phosphorylated nuclear factor kappa B (p-NF-κB) (P < 0.05). In addition, compared with the CON group, the results of flow cytometry and dansylcadaverine (MDC) staining showed a significant increase in apoptosis and autophagy in the EMB-exposed group (P < 0.05) with the activation of the B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X (Bax)/cysteine-aspartic acid protease 3 (Caspase-3)/cysteine-aspartic acid protease 9 (Caspase-9) pathway and microtubule-associated protein light chain 3 (LC3)/sequestosome 1 (p62)/Beclin1 pathway. EMB exposure significantly increased the mRNA and protein expression of receptor-interacting protein 1 (RIPK1)/receptor-interacting protein 3 (RIPK3)/mixed the lineage kinase domain-like (MLKL) pathway (P < 0.05). Moreover, EMB exposure significantly increased the expression of genes related to immunity (immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin D (IgD), and antimicrobial peptide-related genes expression including ß-defensin and hepcidin) (P < 0.05). The addition of RES significantly diminished autophagy, apoptosis, necroptosis, and immunity-related gene expression by inhibiting iNOS activity, NO content, and the protein expression of iNOS and p-NF-κB. In conclusion, RES attenuated autophagy, apoptosis, and necroptosis in EMB-exposed L8824 cells via suppression of the NO system/NF-κB signaling pathway.

Studies on immunotoxicity induced by emamectin benzoate in zebrafish embryos based on metabolomics.

Emamectin benzoate (EMB) is an insecticide for the control of agricultural lepidoptera pests, and also an anti-parasiticide for the control of exoparasites in aquaculture industry. Increased studies suggest that EMB could cause toxicity to non-targeted organisms, but its immunotoxicity to human remains unclear. In this study, zebrafish were used to investigate the immunotoxic effects induced by environmentally relevant doses of EMB. We observed that EMB exposure led to embryo mortality and delayed hatching, as well as increased malformations. Meanwhile, zebrafish exposed to EMB exhibited a significant decrease in the number of neutrophils and macrophages. In addition, untargeted metabolomics approach was developed to elucidate the mechanism of EMB-induced immunotoxicity. We found that a total of 10 shared biomarkers were identified in response to EMB exposure. Furthermore, pathway analysis identified glycerophospholipid metabolism was the most relevant pathway. Within this pathway, it was observed abnormal increases in glycerol 3-phosphate content, which could be attributed to the increased expression of GK5 and decreased expression of GPAT3. Our study provided novel and robust perspectives, which showed that EMB exposure to zebrafish embryos could cause metabolic disturbances that adversely affected development and immune system.

Resveratrol improves emamectin benzoate-induced pyroptosis and inflammation of Ctenopharyngodon idellus hepatic cells by alleviating oxidative stress/endoplasmic reticulum stress.

Emamectin benzoate (EMB) is the most widely used pesticide in the world and contributes to water pollution. Owing to the lack of a specific antidote, EMB has a severe negative impact on the health of aquatic organisms. Resveratrol (RES), a substance with antioxidant capacity, is secreted by the fruits of many plants. This study was to explore the protection of RES against EMB-induced pyroptosis and inflammatory response in grass carp (Ctenopharyngodon idellus) hepatic liver (L8824) cells by oxidative stress/endoplasmic reticulum (ER) stress. The results showed that compared to the CON group, EMB induced oxidative stress in L8824 cells with the increase of reactive oxygen species (ROS), methane dicarboxylic aldehyde (MDA), and hydrogen peroxide (H2O2) contents and the decrease of total superoxide dismutase (t-sod) and glutathione peroxidase (gsh-px) activities (P < 0.05). In addition, EMB triggered ERS, increasing the relative mRNA expression of protein kinase R-like endoplasmic reticulum kinase (perk), inositol requiring enzyme 1 alpha (ire1α), glucose-regulated protein 78 (grp78), activating transcription factor 4 (atf4), activating transcription factor 6 (atf6), and CCAAT-enhancer-binding protein homologous protein (chop) and the protein expression of eukaryotic initiation factor 2α (eif2α), chop, atf6, and atf4. Meanwhile, EMB further induced pyroptosis by upregulating the mRNA and protein expression of nlrp3, aptamer protein (asc), caspase-1, gsdmd, interleukin-1ß (il-1ß), and interleukin-18 (il-18). EMB also induced inflammation in L8824 cells by increasing the mRNA expression of interleukin-2 (il-2), interleukin-6 (il-6), tumor necrosis factor-α (tnf-α), and ifn-γ and decreasing the content of interleukin-10 (il-10). However, compared to the EMB group, the oxidant indices and expression of genes related to ER stress, pyroptosis, and pro-inflammatory factors were significantly down-regulated (P < 0.05), whereas the antioxidant indicators and anti-inflammatory factor were significantly up-regulated in the EMB + RES group (P < 0.05). In conclusion, EMB caused hepatocytes pyroptosis and inflammation in grass carp, and RES could alleviate EMB-induced pyroptosis and inflammation in L8824 cells by ameliorating oxidative stress/ER stress.

Incorporating Bioaccessibility into Inhalation Exposure Assessment of Emamectin Benzoate from Field Spraying.

The inhalation exposure of pesticide applicators and residents who live close to pesticide-treated fields is a worldwide concern in public health. Quantitative assessment of exposure to pesticide inhalation health risk highlights the need to accurately assess the bioaccessibility rather than the total content in ambient air. Herein, we developed an in vitro method to estimate the inhalation bioaccessibility of emamectin benzoate and validated its applicability using a rat plasma pharmacokinetic bioassay. Emamectin benzoate was extracted using the Gamble solution, with an optimized solid-to-liquid ratio (1/250), extraction time (24 h), and agitation (200 rpm), which obtained in vitro inhalation bioaccessibility consistent with its inhalation bioavailability in vivo (32.33%). The margin of exposure (MOE) was used to assess inhalation exposure risk. The inhalation unit exposures to emamectin benzoate of applicators and residents were 11.05-28.04 and 0.02-0.04 ng/m3, respectively, varying markedly according to the methods of application, e.g., formulations and nozzles. The inhalation risk assessment using present application methods appeared to be acceptable; however, the MOE of emamectin benzoate might be overestimated by 32% without considering inhalation bioaccessibility. Collectively, our findings contribute insights into the assessment of pesticide inhalation exposure based on bioaccessibility and provide guidance for the safe application of pesticides.

Residue levels of emamectin benzoate, imidacloprid and its major metabolites in cabbage from 12 regions in China and dietary risk assessment.

A modified quick, easy, cheap, effective, rugged and safe method combined with high-performance liquid chromatography-tandem mass spectrometry was established to investigate the residues of emamectin benzoate (EB), imidacloprid (IMI) and five IMI metabolites (IMI-olefin, IMI-urea, IMI-guanidine, 5-OH and 6-CNA) in cabbage. The average recoveries of the seven compounds in cabbage were 80-102%, with relative standard deviations < 8.0%. The limit of quantification for each compound was 0.01 mg kg-1 . Standardized residue tests were carried out in 12 areas of China under Good Agricultural Practice conditions. A 10% EB-IMI microcapsule suspension was applied once with the high recommended dosage (18 g a.i. ha-1 ) on cabbage. The residues of EB (<0.01 mg kg-1 ), IMI (<0.016 mg kg-1 ) and the sum of IMI and its metabolites (<0.068 mg kg-1 ) in cabbage with the recommended preharvest interval (7 days) were lower than the maximum residue limits in China. Based on residual data from fields, toxicology data and Chinese dietary patterns, dietary risk assessments were conducted. Both the chronic risk quotients (25.2-73.1%) and acute risk quotients (0.43-1.57%) of EB and IMI were below 100%, indicating no unacceptable public health risk for different populations. This study provides guidance on the rational application of these insecticides in cabbage.

Biochemical alterations in cotton leafworm, Spodoptera littoralis (Boisd.) related to emamectin benzoate and fipronil compared to their joint action.

Cotton leafworm, Spodoptera littoralis (Boisduval), is one of the major destructive pests of ornamental, industrial, and vegetable crops. The efficacy of technical emamectin benzoate (EMB) and fipronil (FPR) was assessed against the 4th larval instar using leaf-dip bioassay method. EMB was more efficient than FPR based on 96 h LC50 values of 0.004 and 0.023 µg/ml, respectively. Joint toxic action of the dual exposure in sequence with time interval 24 h and in mix were evaluated at LC10:LC10, LC25:LC25 and LC50:LC50 after 96 h posttreatment, as well. Their impacts on detoxification enzymes, esterases (ESTs); alkaline phosphatase (ALP); and glutathione S-transferase (GST) as well as acetylcholine esterase (AChE) were also determined. The sequential exposure of EMB after FPR (S1) produced antagonism, potentiation, and potentiation effects, respectively while sequential exposure of FPR after EMB (S2) interacted as addition, potentiation, and potentiation respectively. The rest of binary mixtures (Mix) revealed antagonistic effect regardless of concentration. Orthogonal contrast analysis showed that the highest elevations of AChE, α-EST, ß- EST and ALP enzymes were obtained from Mix at LC50:LC50 (181.6%, 288.4, 229.2 and 460.9%, respectively), LC25:LC25 (131.5%, 252.8, 205.60 and 252.0, respectively) and LC10:LC10 (106.6%, 215.6%, 201.8% and 170.0%, respectively). Differently, the greatest elevation of GST activity (157.7%) resulted from S1 at LC50:LC50, while it was significantly lower at LC25:LC25 and LC10:LC10 as well as Mix and S2 at all concentrations than corresponding concentrations of FPR. These findings shed some light on the role of GST in FPR toxicity and clarified the risk of these dual exposures in elevating detoxification enzymes dangerously compared to their individual insecticides. These dual exposures should be carefully handled. Although rotational exposure at low concentrations may enhance performance and mitigate resistance risk, rotational exposure at high concentrations and Mix may indirectly contribute to the evolution of cross-resistance to other insecticides.

Knockdown of the glutamate-gated chloride channel gene decreases emamectin benzoate susceptibility in the fall armyworm, Spodoptera frugiperda.

Emamectin benzoate (EB), a derivative of avermectin, is the primary insecticide used to control the fall armyworm (FAW) in China. However, the specific molecular targets of EB against FAW remain unclear. In this study, we cloned the glutamate-gated chloride channel (GluCl) gene, which is known to be a primary molecular target for avermectin. We first investigated the transcript levels of SfGluCl in FAW and found that the expression level of SfGluCl in the head and nerve cord was significantly higher than that in other tissues. Furthermore, we found that the expression level of SfGluCl was significantly higher in eggs than that in other developmental stages, including larvae, pupae, and adults. Additionally, we identified three variable splice forms of SfGluCl in exons 3 and 9 and found that their splice frequencies remained unaffected by treatment with the LC50 of EB. RNAi mediated knockdown of SfGluCl showed a significant reduction of 42% and 65% after 48 and 72 h of dsRNA feeding, respectively. Importantly, knockdown of SfGluCl sifgnificantly reduced LC50 and LC90 EB treatment induced mortality of FAW larvae by 15% and 44%, respectively, compared to the control group feeding by dsEGFP. In contrast, there were no significant changes in the mortality of FAW larvae treated with the control insecticides chlorantraniliprole and spinetoram. Finally, molecular docking simulations revealed that EB bound to the large amino-terminal extracellular domain of SfGluCl by forming five hydrogen bonds, two alkyl hydrophobic interactions and one salt bridge. These findings strongly suggest that GluCl may serve as one of the molecular targets of EB in FAW, shedding light on the mode of action of this important insecticide.

Exposure to emamectin benzoate confers cytotoxic effects on human molt-4 T-cells and possible ameliorative role of vitamin E and dithiothreitol.

Emamectin benzoate (EMB) is an avermectin insecticide that is extensively used for pest control, but there are few reports concerning its cytotoxic effects on human lymphocytes. In the current study, the hematotoxicity of EMB was evaluated in Molt-4 T-cells, a human T-lymphoblastic cell line with high motility, and the role of vitamin E (VitE) and dithiothreitol (DTT) in attenuating EMB cytotoxicity was characterized. Exposure of Molt-4 cells to EMB decreased cell viability and proliferation, induced a loss of cell clusters, and significantly increased membrane collapse and chromatin condensation. Moreover, EMB significantly increased cell death and suppressed transglutaminase activity. EMB treatment modulated the NF-κB signaling pathway, decreased the expression of p105, p50, and p65/RelA in cytosolic and nuclear fractions, and increased nuclear IκBα expression. EMB increased oxidative stress, as demonstrated by a significant increase in the levels of reactive oxygen species (ROS). Treatment with non-cytotoxic concentrations of VitE or DTT ameliorated the hematotoxicity induced by pretreatment with EMB, increased Molt-4 cell viability, raised the IC50 values of EMB, limited intracellular ROS generation, and mitigated EMB-mediated effects on NF-κB signaling. The results indicate the potential cytotoxicity of EMB on human lymphocytes, and demonstrate that VitE and DTT treatment can reduce the cytotoxic effects of EMB.

The potential immunotoxicity of emamectin benzoate on the human THP-1 macrophages.

Emamectin benzoate (EMB) as one of the typical biological pesticides has a wide range of applications in agriculture. However, the immune toxic effects of EMB in human received limited attention. In our study, THP-1 macrophage as an in vitro model was used to evaluate immune functions exposed to EMB. We observed that EMB inhibited phagocytic activity and respiratory burst capacity of macrophages without inducing cellular toxicity, implying the potential immunosuppression. Besides, EMB disturbed the cytokines balance embodied in the increase of TNF-α, IL-1ß, IL-6, CCL27, CXCL8 mRNA expression and the decrease of IL-4, IL-13, IL-10 mRNA expression. EMB could exhibit pro-inflammatory responses in macrophages and promote the conversion of macrophages to M1 phenotype. Moreover, NF-κB pathway involved in regulating immune function from KEGG pathway analysis. EMB exposure could activate the NF-κB pathway in THP-1 macrophages by exploring the critical proteins. This research provided insights on immunotoxicity evaluation and clarified EMB-induced immunotoxicity was related to NF-κB pathway activation.

Investigation on the immunotoxicity induced by Emamectin benzoate on THP-1 macrophages based on metabolomics analysis.

Emamectin benzoate (EMB) is an insecticide extensively used in agricultural area. Assessing the toxic effects of EMB in mammals or humans and its endogenous metabolites alteration are the appropriate means of evaluating its risks to human health. In the study, THP-1 macrophage, a human immune model, was applied to investigate the immunotoxicity of EMB. A global metabolomics approach was developed to analyze metabolic perturbation on macrophages and discover the potential biomarkers of EMB-induced immunotoxicity. The results indicated that EMB could inhibit immune functions of macrophages. Based on metabolomics analysis, our results illustrated that EMB caused significant alterations in metabolic profiles on macrophages. 22 biomarkers associated with immune response were screened by pattern recognition and multivariate statistical analysis. Furthermore, pathway analysis identified purine metabolism was the most relevant pathway in the metabolic process and the abnormal conversion of AMP to xanthosine regulated by NT5E might be a potential mechanism of immunotoxicity induced by EMB. Our study provides important insights for understanding and underlying mechanism of immunotoxicity exposed to EMB.

Neurodevelopmental Toxicity of Emamectin Benzoate to the Early Life Stage of Zebrafish Larvae (Danio rerio).

Emamectin benzoate (EMB) is a widely used pesticide and feed additive in agriculture and aquaculture. It easily enters the aquatic environment through various pathways, thus causing adverse effects on aquatic organisms. However, there are no systematic studies regarding the effects of EMB on the developmental neurotoxicity of aquatic organisms. Therefore, the aim of this study was to evaluate the neurotoxic effects and mechanisms of EMB at different concentrations (0.1, 0.25, 0.5, 1, 2, 4 and 8 µg/mL) using zebrafish as a model. The results showed that EMB significantly inhibited the hatching rate, spontaneous movement, body length, and swim bladder development of zebrafish embryos, as well as significantly increased the malformation rate of zebrafish larvae. In addition, EMB adversely affected the axon length of motor neurons in Tg (hb9: eGFP) zebrafish and central nervous system (CNS) neurons in Tg (HuC: eGFP) zebrafish and significantly inhibited the locomotor behavior of zebrafish larvae. Meanwhile, EMB induced oxidative damage and was accompanied by increasing reactive oxygen species in the brains of zebrafish larvae. In addition, gene expression involvement in oxidative stress-related (cat, sod and Cu/Zn-sod), GABA neural pathway-related (gat1, gabra1, gad1b, abat and glsa), neurodevelopmental-related (syn2a, gfap, elavl3, shha, gap43 and Nrd) and swim bladder development-related (foxa3, pbxla, mnx1, has2 and elovlla) genes was significantly affected by EMB exposure. In conclusion, our study shows that exposure to EMB during the early life stages of zebrafish significantly increases oxidative damage and inhibits early central neuronal development, motor neuron axon growth and swim bladder development, ultimately leading to neurobehavioral changes in juvenile zebrafish.

Function of Cytochrome P450s and Gut Microbiome in Biopesticide Adaptation of Grapholita molesta on Different Host Diets.

Insects that feed on various host plants possess diverse xenobiotic adaptations; however, the underlying mechanisms are poorly understood. In the present study, we used Grapholita molesta, which shifts feeding sites from peach shoots to apple fruits, as a model to explore the effects of shifts in host plant diet on the profiles of cytochrome P450s and the gut bacteria microbiome, as well as their effects on biopesticide adaptation. We found that the sensitivity of the fruit-feeding G. molesta to emamectin benzoate biopesticide was significantly lower than that of the shoot-feeding larvae. We also found that the P450 enzyme activity and the expression of nine cytochrome P450s were enhanced in G. molesta fed on Fuji apples compared to those fed on peach shoots. The survival rates of G. molesta exposed to emamectin benzoate significantly decreased as each of three of four emamectin benzoate-inducted cytochrome P450 genes were silenced. Furthermore, we discovered the gut bacteria dynamics of G. molesta changed with the host shift and the structure of the gut bacteria microbiome was determined by the final diet ingested; additionally, the dysbiosis of the gut microbiota induced by antibiotics could significantly increase the sensitivity to emamectin benzoate. Taken together, our results suggest that the expression of P450s and the composition of the gut bacteria microbiome promote adaptation to emamectin benzoate in G. molesta, providing new insights into the molecular mechanisms underlying xenobiotic adaptation in this notorious pest.

Dissipation Dynamic, Residue Distribution and Risk Assessment of Emamectin Benzoate in Longan by High-Performance Liquid Chromatography with Fluorescence Detection.

A derivatization method combined with high-performance liquid chromatography-fluorescence detection (HPLC-FLD) was used to evaluate the dissipation, residue distribution and risk assessment of emamectin benzoate in whole longan and pulp. The average recoveries were 82-111% with relative standard deviation (RSD) less than 11%. The limit of quantification (LOQ) was 0.001 mg/kg in longan and pulp. The half-lives were 3.3-4.2 days. The terminal residues in whole longan were <0.001-0.025 mg/kg applied two and three times at two levels of dosage with PHIs of 10, 14, and 21 days. The residues in whole longan had a higher quantity than those in the pulp, and the terminal residues of pulp were all lower than LOQ (0.001 mg/kg). The chronic risk of emamectin benzoate was not negligible to humans depending on ADI% value, which was higher than 1; and the acute risk was acceptable to the consumer. This study could provide guidance for the safe use of emamectin benzoate in longan and serve as a reference for the establishment of maximum residue limits (MRLs) in China.

Enhancement of photocatalytic activity of Ba-doped CoO for degradation of Emamectin benzoate in aqueous solution.

The present study was focused on the preparation of cobalt oxide (CoO) and barium-doped cobalt oxide (Ba-doped CoO) by following the co-precipitation method for the degradation of Emamectin benzoate pesticide in the aqueous medium. The prepared catalysts were characterized using SEM, EDX, and XRD to confirm the formation of catalysts and to observe the variation in the composition of catalysts during the degradation study. It can be suggested from the results of SEM, EDX, XRD, and FTIR analyses that Ba atom has successfully incorporated in the crystalline structure of CoO. The degradation of Emamectin benzoate pesticide was studied under the influence of different factors like solution pH, the dose of catalyst, contact time, temperature, and initial concentration of pesticide. It was observed that solution pH affects the degradation of the pesticide, and maximum degradation (23% and 54%) was found at pH 5.0 and 6.0 using CoO and Ba-doped CoO, respectively. The degradation of pesticides was found to be increased continuously (27-35% in case of CoO while 47-58% in case Ba-doped CoO) with the time of contact. However, the degradation was found to be decreased (23-3% in case of CoO while 47-44% in case Ba-doped CoO) with an increase in temperature. Likewise, in the beginning, degradation was observed to be increased up to some extent with the dose of catalyst and initial concentration of pesticide but started to decrease with further augmentation in the dose of catalyst and initial concentration of pesticide. It may be concluded from this study that doping of Ba considerably enhanced the photocatalytic ability of CoO for Emamectin benzoate pesticide.

The Impact of Magnetic Field on Insecticide Toxicity Measured by Biological and Biochemical Parameters of Earias insulana (Boisd).

This study illustrates the effect of magnetic field (MF) on the toxicity of two insecticides, emamectin benzoate (Emazoate 2.15% EC) and spinosad (SpinTor 24% SC), and determines their adverse effects on the bollworm (Earias insulana) through various biological and biochemical assays. The investigation indicated that exposure to the insecticides in a MF of 180 mT resulted in stronger toxicity, with LC50 values of 0.162, 1.211, and 1.770 ppm, respectively. In addition, the results showed that magnetized insecticides significantly increased in the duration of the total immature stages (larvae and/or pupae) 32.1 and 36.6 days, compared with 27.9 and 30.5 days, respectively, in the nonmagnetized insecticides, while untreated check was 21 days. Also, the magnetized insecticides reduced the percentage of adult emergence, and increased deformations in the larval and pupal stages. Furthermore, sex ratio was greatly affected by exposure to both insecticides in conjunction with the MF. Exposure of the larvae of E insulana to magnetized insecticides can bring about malfunction in some biochemical process and significantly decreased the invertase activity, and decreased the total protein and carbohydrates. In contrast, it can increase amylase compared with nonmagnetized insecticides and untreated controls. Results concluded that the two insecticides' MF affected growth, survival time, and biological and biochemical parameters of E. insulana. © 2022 Bioelectromagnetics Society.

Lethal and sublethal effects in Pink salmon (Oncorhynchus gorbuscha) following exposure to five aquaculture chemotherapeutants.

Early life stages of Pink salmon (Oncorhynchus gorbuscha) are at risk of exposure to the active ingredients of chemotherapeutant formulations (hydrogen peroxide [HP], azamethiphos [AZ], emamectin benzoate [EB], cypermethrin [CP] and deltamethrin [DM]) used to control sea lice in salmon aquaculture. LC50 values (95% confidence intervals) for acute 48-h water exposures in order of least to most toxic to seawater-adapted pink salmon fry were: HP (227 [138-418] mg/L), EB (1090 [676-2006] µg/L), AZ (80 [52-161] µg/L), CP (5.1 [3.0-10.5] µg/L), and DM (980 [640-1800] ng/L), and in subchronic 10-d lethality sediment exposure tests: EB (2065 [1384-3720] µg/kg), CP (97 [58-190] µg/kg), and DM (1035 [640-2000] ng/kg). Alterations in behaviour varied between chemicals; no chemical attracted pink salmon fry; fish avoided HP to a limited extent at 50 mg/L), as well as EB (300 µg/L), and AZ (50 µg/L). Significant concentration-dependent decreases in olfactory responsiveness to food extract were seen following AZ, CP and DM exposures that occurred at lower concentrations with longer exposure periods (10 µg/L, 0.5 µg/L and 100 ng/L thresholds at 7 d). Following 10-d sediment exposures, olfaction was only affected by CP exposure at 50 µg/kg. Significant decreases in swimming performance (Ucrit) occured for HP, AZ, CP and DM at concentrations as low as 100 mg/L, 10 µg/L, 2 µg/L and 200 ng/L, respectively. This study provides comprehensive data on the lethal and sublethal effects of aquaculture chemotherapeutant exposure in early life stage pink salmon.

Toxic evaluation of Proclaim Fit® on adult and larval worker honey bees.

Impacts to honey bees due to exposure to agricultural pesticides is one of the most serious threats to the beekeeping industry. Our research evaluated toxicity of the formulated insecticides Lufenuron+Emamectin benzoate (Proclaim Fit®) on the European honey bee Apis mellifera L. at field-realistic concentration (worst-case scenario). Newly emerged (≤24-h old) and forager (unknown age) worker bees were treated with the field recommended concentration of Proclaim Fit® using three routes of exposure including residual contact, oral, and spray within the laboratory. We also assessed the effects of Proclaim Fit® on the specific activity of some well-known detoxifying enzymes including α-esterase, ß-esterase, and Glutathione S-transferase (GST) in the honey bees. In addition, toxicity of the formulation was tested on 4th instar larvae within the hive. Based on estimated median survival times (MSTs), Proclaim Fit® was highly toxic to the bees, especially when applied as spray. According to our estimated relative median potency (RMP) values, newly emerged bees were 1.72× more susceptible than foragers to Proclaim Fit® applied orally. Enzyme assays revealed the considerable involvement of the enzymes, especially GST and α-esterase, in detoxification of the Proclaim Fit®, but their activities were significantly influenced by route of exposure and age of bee. Notably, Proclaim Fit® was highly toxic to 4th instar honey bee larvae. Our results generally indicate a potent toxicity of Proclaim Fit® toward honey bees. Therefore, its application requires serious consideration and adherence to strict guidelines, especially during the flowering time of crops.

High frequency of ryanodine receptor and cytochrome P450 CYP9A186 mutations in insecticide-resistant field populations of Spodoptera exigua from China.

The beet armyworm, Spodoptera exigua is a global agricultural pest that is polyphagous, highly dispersive, and often difficult to control due to resistance to many insecticides. Previous studies showed that a target site mutation in the S. exigua ryanodine receptor (SeRyR) corresponding to I4743M contributes approximately 20-fold resistance to chlorantraniliprole, whereas a mutation in the cytochrome P450 enzyme CYP9A186 corresponding to F116V confers 200-fold to emamectin benzoate through enhanced metabolic detoxification. Here, high frequencies of mutations were found among six China S. exigua field populations collected from 2016 to 2019 resulting in SeRyR I4743M and CYP9A186 F116V substitutions, with some populations having high levels of resistance to chlorantraniliprole and emamectin benzoate, respectively. Whereas we found a significant correlation between emamectin benzoate resistance level and the allele frequency of CYP9A186 F116V, no significant correlation was found between chlorantraniliprole resistance level and SeRyR I4743M allele frequency in the six field populations. These results suggest that CYP9A186 F116V is a major resistance mechanism for emamectin benzoate in the tested field populations, whereas it is likely that resistance mechanisms other than SeRyR I4743M are responsible for resistance to chlorantraniliprole in the six China field populations. Because of the growing resistance to these two insecticides by S. exigua in China, the use of insecticidal compounds with different modes of action and/or other integrated pest management strategies are needed to further delay the evolution of insecticide resistance and effectively manage S. exigua in China.

Expression stability of candidate RT-qPCR housekeeping genes in Spodoptera frugiperda (Lepidoptera: Noctuidae).

Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is commonly used to quantify gene expression. For normalization, the expression of each gene is compared with a reference "housekeeping" gene that is stably expressed under relevant stress. Unfortunately, there have been no reports on the stability of such reference genes under various treatments of the Spodoptera frugiperda. In this study, we used five tools (RefFinder, GeNorm, NormFinder, BestKeeper, and ΔCt methods) to evaluate the stability of 12 candidate reference genes (RPS18, ß-tubulin, GAPDH, RPS7, RPS15, RPL7, RPL32, Actin-5C, EF1-α, EF1-γ, RPL27, and ACE) in different instars, tissues, and treatments (high and low temperature, UV-A, and emamectin benzoate). Several ribosomal proteins (RPS7, RPS15, RPL32, RPS18, and RPL7), GAPDH, Actin-5C, and ß-tubulin, were relatively stable, suggesting that they are ideal housekeeping genes for various treatments. ACE was extremely unstable under various experimental treatments, rendering it unsuitable as an internal reference. This study identified the reference housekeeping genes stably expressed by S. frugiperda under different treatments, thus setting a foundation for further exploration of the physiological and biochemical mechanisms.