Multiple stresses induced by chronic exposure to flupyradifurone affect honey bee physiological states.

Flupyradifurone (FPF) has been reported to have a potential risk to terrestrial and aquatic ecosystems. In the present study, the effects of chronic FPF exposure on bees were systematically investigated at the individual behavioral, tissue, cell, enzyme activity, and the gene expression levels. Chronic exposure (14 d) to FPF led to reduced survival (12 mg/L), body weight gain (4 and 12 mg/L), and food utilization efficiency (4 and 12 mg/L). Additionally, FPF exposure (12 mg/L) impaired sucrose sensitivity and memory of bees. Morphological analysis revealed significant cellular and subcellular changes in brain neurons and midgut epithelial cells, including mitochondrial damage, nuclear disintegration, and apoptosis. FPF exposure (4 and 12 mg/L) led to oxidative stress, as evidenced by increased lipid peroxidation and alterations in antioxidant enzyme activity. Notably, gene expression analysis indicated significant dysregulation of apoptosis, immune, detoxification, sucrose responsiveness and memory-related genes, suggesting the involvement of different pathways in FPF-induced toxicity. The multiple stresses and potential mechanisms described here provide a basis for determining the intrinsic toxicity of FPF.

Interaction of acetamiprid, Varroa destructor, and Nosema ceranae in honey bees.

Health of honey bees is threatened by a variety of stressors, including pesticides and parasites. Here, we investigated effects of acetamiprid, Varroa destructor, and Nosema ceranae, which act either alone or in combination. Our results suggested that interaction between the three factors was additive, with survival risk increasing as the number of stressors increased. Although exposure to 150 µg/L acetamiprid alone did not negatively impact honey bee survival, it caused severe damage to midgut tissue. Among the three stressors, V. destructor posed the greatest threat to honey bee survival, and N. ceranae exacerbated intestinal damage and increased thickness of the midgut wall. Transcriptomic analysis indicated that different combinations of stressors elicited specific gene expression responses in honey bees, and genes involved in energy metabolism, immunity, and detoxification were altered in response to multiple stressor combinations. Additionally, genes associated with Toll and Imd signalling, tyrosine metabolism, and phototransduction pathway were significantly suppressed in response to different combinations of multiple stressors. This study enhances our understanding of the adaptation mechanisms to multiple stressors and aids in development of suitable protective measures for honey bees. ENVIRONMENTAL IMPLICATION: We believe our study is environmentally relevant for the following reasons: This study investigates combined effects of pesticide, Varroa destructor, and Nosema ceranae. These stressors are known to pose a threat to long-term survival of honey bees (Apis mellifera) and stability of the ecosystems. The research provides valuable insights into the adaptive mechanisms of honey bees in response to multiple stressors and developing effective conservation strategies. Further research can identify traits that promote honey bee survival in the face of future challenges from multiple stressors to maintain the overall stability of environment.

Interactive effects of dinotefuran and Nosema ceranae on the survival status and gut microbial community of honey bees.

Growing evidences have shown that the decline in honey bee populations is mainly caused by the combination of multiple stressors. However, the impacts of parasitic Nosema ceranae to host fitness during long-term pesticide exposure-induced stress is largely unknown. In this study, the effects of chronic exposure to a sublethal dose of dinotefuran, in the presence or absence of N. ceranae, was examined in terms of survival, food consumption, detoxification enzyme activities and gut microbial community. The interaction between dinotefuran and Nosema ceranae on the survival of honey bee was synergistic. Co-exposure to dinotefuran and N. ceranae led to less food consumption and greater changes of enzyme activities involved in defenses against oxidative stress. Particularly, N. ceranae and dinotefuran-N. ceranae co-exposure significantly impacted the gut microbiota structure and richness in adult honey bees, while dinotefuran alone did not show significant alternation of core gut microbiota compared to the control group. We herein demonstrated that chronical exposure to dinotefuran decreases honey bee's survival but is not steadily associated with the gut microbiota dysbiosis; by contrast, N. ceranae parasitism plays a dominant role in the combination in influencing the gut microbial community of the host honey bee. Our findings provide a comprehensive understanding of combinatorial effects between biotic and abiotic stressors on one of the most important pollinators, honey bees.

Greater wax moth control in apiaries can be improved by combining Bacillus thuringiensis and entrapments.

The greater wax moth (GWM), Galleria mellonella (Lepidoptera: Pyralidae), is a major bee pest that causes significant damage to beehives and results in economic losses. Bacillus thuringiensis (Bt) appears as a potential sustainable solution to control this pest. Here, we develop a novel Bt strain (designated BiotGm) that exhibits insecticidal activity against GWM larvae with a LC50 value lower than 2 µg/g, and low toxicity levels to honey bee with a LC50 = 20598.78 µg/mL for larvae and no observed adverse effect concentration = 100 µg/mL for adults. We design an entrapment method consisting of a lure for GWM larvae, BiotGm, and a trapping device that prevents bees from contacting the lure. We find that this method reduces the population of GWM larvae in both laboratory and field trials. Overall, these results provide a promising direction for the application of Bt-based biological control of GWM in beehives, although further optimization remain necessary.

Acute and chronic effects of sublethal neonicotinoid thiacloprid to Asian honey bee (Apis cerana cerana).

Pesticide pollution is one of the most important factors for global bee declines. Despite many studies have revealed that the most important Chinese indigenous species，Apis cerana, is presenting a high risk on exposure to neonicotinoids, the toxicology information on Apis cerana remain limited. This study was aimed to determine the acute and chronic toxic effects of thiacloprid (IUPAC name: {(2Z)-3-[(6-Chloro-3-pyridinyl)methyl]-1,3-thiazolidin-2-ylidene}cyanamide) on behavioral and physiological performance as well as genome-wide transcriptome in A. cerana. We found the 1/5 LC50 of thiacloprid significantly impaired learning and memory abilities after both acute and chronic exposure, nevertheless, has no effects on the sucrose responsiveness and phototaxis climbing ability of A. cerana. Moreover, activities of detoxification enzyme P450 monooxygenases and CarE were increased by short-term exposure to thiacloprid, while prolonged exposure caused suppression of CarE activity. Neither acute nor chronic exposure to thiacloprid altered honey bee AChE activities. To further study the potential defense molecular mechanisms in Asian honey bee under pesticide stress, we analyzed the transcriptomes of honeybees in response to thiacloprid stress. The transcriptomic profiles revealed consistent upregulation of immune- and stress-related genes by both acute or chronic treatments. Our results suggest that the chronic exposure to thiacloprid produced greater toxic effects than a single administration to A. cerana. Altogether, our study deepens the understanding of the toxicological characteristic of A. cerana against thiacloprid, and could be used to further investigate the complex molecular mechanisms in Asian honey bee under pesticide stress.

Interaction of chlorothalonil and Varroa destructor on immature honey bees rearing in vitro.

Under realistic environmental conditions, bees are often exposed to multiple stressors, especially Varroa destructor and pesticides. In this study, the effects of exposure to NOAEC of chlorothalonil during the larval stage, in the presence or absence of V. destructor, was examined in terms of survival, morphological and transcriptional changes. The interaction between chlorothalonil and V. destructor on the survival of honey bee was additive. V. destructor are the dominant factor in the interaction for survival and transcriptome alternation. The downregulation of the genes related to tissue growth and caste differentiation may directly link to the mortality of honey bees. Either chlorothalonil or V. destructor induces the irregular morphology of trophocytes and oenocytes in the fat body. In addition to irregular shapes, oenocytes in V. destructor alone and double-stressor treatment group showed altered nuclei and vacuoles in the cytoplasm. The interaction of V. destructor and chlorothalonil at the larval stage have potential adverse effects on the subsequent adult bees, with up-regulation of genes involved in lipid metabolism and detoxification/defense in fat body tissue. Our findings provide a comprehensive understanding of combinatorial effects between biotic and abiotic stressors on one of the most important pollinators, honey bees.

Chronic larval exposure to thiacloprid impairs honeybee antennal selectivity, learning and memory performances.

The use of agricultural neonicotinoid insecticides has sub-lethal chronic effects on bees that are more prevalent than acute toxicity. Among these insecticides, thiacloprid, a commonly used compound with low toxicity, has attracted significant attention due to its potential impact on the olfactory and learning abilities of honeybees. The effect of sub-lethal larval exposure to thiacloprid on the antennal activity of adult honeybees (Apis mellifera L.) is not yet fully understood. To address this knowledge gap, laboratory-based experiments were conducted in which honeybee larvae were administered thiacloprid (0.5 mg/L and 1.0 mg/L). Using electroantennography (EAG), the impacts of thiacloprid exposure on the antennal selectivity to common floral volatiles were evaluated. Additionally, the effects of sub-lethal exposure on odor-related learning and memory were also assessed. The results of this study reveal, for the first time, that sub-lethal larval exposure to thiacloprid decreased honeybee antenna EAG responses to floral scents, leading to increased olfactory selectivity in the high-dose (1.0 mg/L) group compared to the control group (0 mg/L vs. 1.0 mg/L: p = 0.042). The results also suggest that thiacloprid negatively affected odor-associated paired learning acquisition, as well as medium-term (1 h) (0 mg/L vs. 1.0 mg/L: p = 0.019) and long-term memory (24 h) (0 mg/L vs. 1.0 mg/L: p = 0.037) in adult honeybees. EAG amplitudes were dramatically reduced following R-linalool paired olfactory training (0 mg/L vs. 1.0 mg/L: p = 0.001; 0 mg/L vs. 0.5 mg/L: p = 0.027), while antennal activities only differed significantly in the control between paired and unpaired groups. Our results indicated that exposure to sub-lethal concentrations of thiacloprid may affect olfactory perception and learning and memory behaviors in honeybees. These findings have important implications for the safe use of agrochemicals in the environment.

Thiacloprid impairs honeybee worker learning and memory with inducing neuronal apoptosis and downregulating memory-related genes.

Neonicotinoids are among the most widely used insecticides in the world and are recognized as a potential cause of pollinator decline. Previous studies have demonstrated that the neonicotinoid thiacloprid has adverse effects on foraging and memory behaviors. However, there is no direct evidence linking thiacloprid-induced neuronal cell damage in the brains of honeybees to learning and memory dysfunction. Adult honeybee (Apis mellifera L.) workers were chronically exposed to sub-lethal concentrations of thiacloprid. We discovered that thiacloprid negatively affected their survival, food consumption, and body weight. In addition, sucrose sensitivity and memory performance were impaired. We evaluated the apoptosis of honeybee brain cells using TUNEL (Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling) and Caspase-3 assays, which revealed that thiacloprid increases the dose-dependent apoptosis of neurons in the mushroom bodies (MB) and antennal lobes (AL). We also determined the abnormal transcripts of multiple genes, including vitellogenin (Vg), immune system genes (apidaecin and catalase), and memory-associated genes (pka, creb, Nmdar1, Dop2, Oa1, Oa-2R, and Oa-3R). These results indicate that exposure to sublethal concentrations of thiacloprid cause abnormal expression of memory-related genes and apoptosis of brain cells in the AL and MB, which may contribute to the memory disorder induced by thiacloprid exposure.

Acute oral toxicity, apoptosis, and immune response in nurse bees (Apis mellifera) induced by flupyradifurone.

The potential toxicity of flupyradifurone (FPF) to honey bees has been a subject of controversy in recent years. Understanding the effect of pesticides on nurse bees is important because the fitness of nurse bees is critical for in-hive activities, such as larval survival and performing hive maintenance. In order to evaluate the acute oral toxicity of flupyradifurone on nurse bees, flupyradifurone at five different concentrations was selected to feed both larvae and nurse bees. Our results showed that nurse bees were more sensitive to flupyradifurone than larvae (LD50 of the acute oral toxicity of flupyradifurone was 17.72 µg a.i./larva and 3.368 µg a.i./nurse bee). In addition, the apoptotic rates of neurons in mushroom bodies of nurse bees were significantly induced by flupyradifurone at sublethal concentrations (8 mg/L, 20 mg/L, and 50 mg/L) and the median lethal concentration LC50 (125 mg/L). The expression of immune-related genes (Hsp90, Toll-8/Tollo, and defensin) was significantly changed in exposed nurse bees at the field-realistic concentration of flupyradifurone. However, three detoxifying enzyme genes (CYP9Q1, -2, and -3) were not affected by pesticide exposure. Our data suggest that although flupyradifurone had a relatively lower acute oral toxicity than many other common pesticides, exposures to the field-realistic and other sublethal concentrations of flupyradifurone still have cytotoxicity and immune-responsive effects on nurse bees. Therefore, flupyradifurone should be considered for its application in crops.

Exposure of chlorothalonil and acetamiprid reduce the survival and cause multiple internal disturbances in Apis mellifera larvae reared in vitro.

Background: Chlorothalonil and acetamiprid are chemical pesticides commonly used in agricultural production and have been shown to have negative effects on bee's fitness. Despite many studies have revealed that honey bee (Apis mellifera L.) larvae are posting a high risk on exposure to pesticides, but the toxicology information of chlorothalonil and acetamiprid on bee larvae remain limited. Results: The no observed adverse effect concentration (NOAEC) of chlorothalonil and acetamiprid for honey bee larvae were 4 µg/mL and 2 µg/mL, respectively. Except for CarE, the enzymic activities of GST and P450 were not influenced by chlorothalonil at NOAEC, while chronic exposure to acetamiprid slightly increased the activities of the three tested enzymes at NOAEC. Further, the exposed larvae showed significantly higher expression of genes involved in a series of different toxicologically relevant process following, including caste development (Tor (GB44905), InR-2 (GB55425), Hr4 (GB47037), Ac3 (GB11637) and ILP-2 (GB10174)), immune system response (abaecin (GB18323), defensin-1 (GB19392), toll-X4 (GB50418)), and oxidative stress response (P450, GSH, GST, CarE). Conclusion: Our results suggest that the exposure to chlorothalonil and acetamiprid, even at concentrations below the NOAEC, showed potentially effects on bee larvae's fitness, and more important synergistic and behavioral effects that can affect larvae fitness should be explored in the further.

Pesticide exposure and forage shortage in rice cropping system prevents honey bee colony establishment.

As one of the key stable crops to feed half of the world's population, how rice cropping system affects honey bee health regarding pesticide exposure and forage availability is under investigated. We predicted honey bees were stressed by high pesticide exposure and forage dearth in monoculture rice systems. Providing access to natural habitats is a typical approach to mitigate the negative impact of intensive agriculture on honey bees. We aimed to determine if bee colonies located in landscapes with more cover of forest habitat would collect more forage and be exposed to less pesticides. We selected beekeeping locations in rice dominated landscapes (as control), mosaic landscapes of rice and medium woodland (MW) cover, and landscapes of high woodland (HW) cover, respectively, in July when rice starts bloom and pesticides are commonly used. Colonies were inspected at a biweekly frequency from July to October with population growth and forage (nectar and pollen) availability estimated. Pollen and bees were collected in middle August for pesticide exposure analysis. We did not observe enhancement in forage availability and reduction in pesticide exposure in landscapes with increased forest habitat (i.e., MW or HW cover), and all colonies failed in the end. Other natural habitats that can supplement flower shortage periods in forest can be considered for supporting bee health. Our results suggest that forest should be carefully assessed for being incorporated into beekeeping management or pollinator conservation when forest phenology can be a factor to affect its impact as a natural habitat.

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.

Early-Life Sublethal Thiacloprid Exposure to Honey Bee Larvae: Enduring Effects on Adult Bee Cognitive Abilities.

Honey bees have significant ecological and economic value as important pollinators, but they are continuously exposed to various environmental stressors, including insecticides, which can impair their health and cause colony decline. (1) Background: Cognitive abilities are vital for the functional maintenance of honey bees; however, it remains unknown if chronic, low-dose exposure to thiacloprid during the larval stage impairs the cognitive abilities of emerged adult honey bees. (2) Methods: To explore this question, honey bee larvae were fed 0, 0.5, and 1.0 mg/L thiacloprid during their developmental phase. Then, the cognitive (i.e., olfactory learning and memory) abilities of adult honey bees were quantified to assess the delayed impacts of early-stage thiacloprid exposure on adult honey bee cognition. Neural apoptosis and transcriptomic level were also evaluated to explore the neurological mechanisms underlying these effects. (3) Results: Our results revealed that chronic larval exposure to sublethal thiacloprid impaired the learning and memory abilities of adult honey bees by inducing neuronal apoptosis and transcriptomic alterations. (4) Conclusions: We highlighted a previously unknown impairment caused by thiacloprid in honey bees.

Combined transcriptome and metabolite profiling analyses provide insights into the chronic toxicity of carbaryl and acetamiprid to Apis mellifera larvae.

Despite many studies have revealed that developing honey bee (Apis mellifera) larvae are posting a high risk on exposure to insecticides, the toxicology information on bee larvae remain limited. The present study demonstrated the first assessment of the effects of no observed adverse effect concentration (NOAEC) of carbaryl (CR) and acetamiprid (ACE) on transcriptome and metabolome in honeybee larvae reared in vitro. Chronic exposure to carbaryl caused transcriptional disorders associated with oxidative stress. In addition, a series of metabolic homeostasis were disrupted by carbaryl stress, such amino acid metabolism, purine and pyrimidine metabolism and flavone and flavonol biosynthesis. The activities of enzymic biomarkers including GST, P450, CAT, AChE and SOD were not influenced by ACE stress, while the CR exposure slightly decreased the activity of CAT and SOD. Our results clearly show that ACE and CR display different potential to modulate transcriptome and metabolome associated with their different toxicity against bee larvae.

Recovery and genetic characterization of black queen cell virus.

Black queen cell virus (BQCV) is a severe threat to the honeybee (Apis mellifera) worldwide. Although several BQCV strains have been reported in China, the molecular basis for BQCV pathogenicity has not been well understood. Thus, a reverse genetic system of BQCV is required for studying viral replication and its pathogenic mechanism. Here, the complete genome sequence of BQCV was obtained from honeybees using reverse transcription PCR (RT-PCR), namely a BQCV China-GS1 strain (KY741959). Then, a phylogenetic tree was built to analyse the genetic relationships among BQCV strains from different regions. Our results showed that the BQCV China-GS1 contained two ORFs, consistent with the known reference strains, except for the BQCV China-JL1 strain (KP119603). Furthermore, the infectious clone of BQCV was constructed based on BQCV China-GS1 using a low copy vector pACYC177 and gene recombination. Due to the lack of culture cells for bee viruses, we infected the healthy bees with infectious clone of BQCV, and the rescued BQCV resulted in the recovery of recombinant virus, which induced higher mortality than those of the control group. Immune response after inoculated with BQCV further confirmed that the infectious clone of BQCV caused the cellular and humoral immune response of honeybee (A. mellifera). In conclusion, the full nucleotide sequence of BQCV China-GS1 strain was determined, and the infectious clone of BQCV was constructed in this study. These data will improve the understanding of pathogenesis and the host immune responses to viral infection.

Chlorothalonil alters the gut microbiota and reduces the survival of immature honey bees reared in vitro.

BACKGROUND: Chlorothalonil is a nonsystemic fungicide, and it is one of the most widely detected pesticides in bee hives. The effect of chlorothalonil on the survival, weight, and gut microbiota of immature Apis mellifera L. reared in vitro was studied. RESULTS: Larvae were fed 1, 2, 4, 8, and 16 µg/mL chlorothalonil and compared with larvae fed the negative control (diet without any additives), positive control (45 mg/L dimethoate), and solvent control (2% acetone). Compared with the control groups, the survival of the 2, 4, 8, and 16 µg/mL chlorothalonil treatments was significantly reduced. The no-observed-adverse-effect concentration of chlorothalonil was 1 µg/mL. Chlorothalonil had no significant effect on larval weight. The gut bacterial community composition of newly emerged bees was determined by PacBio 16S rDNA gene sequencing. linear discriminant analysis effect size (LEFSe) analysis showed that Pseudomonadales and Burkholderiales were affected by exposure to chlorothalonil. CONCLUSION: Chlorothalonil reduced the survival of honey bee larvae and altered the gut microbiota of newly emerged bees. The risk of pesticides to honey bees is related to their toxicity and exposure dose.

Tropilaelaps mercedesae parasitism changes behavior and gene expression in honey bee workers.

Tropilaelaps mercedesae is one of the most problematic honey bee parasites and has become more threatening to the beekeeping industry. Tropilaelaps can easily parasitize immature honey bees (larvae and pupae) and have both lethal and sublethal effects on the individual worker bees. Our study for the first time experimentally assessed the effects of T. mercedesae on olfactory learning, flight ability, homing ability as well as transcriptional changes in parasitized adult honey bees. T. mercedesae infestation had negative impacts on olfactory associated function, flight ability, and homing rate. The volume of the mushroom body significantly increased in infested honey bees, which may be correlated to the lower sucrose responsiveness as well as lower learning ability in the infested bees. The gene expression involved in immune systems and carbohydrate transport and metabolism were significantly different between infested bees and non-infested bees. Moreover, genes function in cell adhesion play an essential role in olfactory sensory in honey bees. Our findings provide a comprehensive understanding of European honey bees in response to T. mercedesae infestation, and could be used to further investigate the complex molecular mechanisms in honey bees under parasitic stress.

The Effects of Exposure to Flupyradifurone on Survival, Development, and Foraging Activity of Honey Bees (Apis mellifera L.) under Field Conditions.

Flupyradifurone (FPF) is a novel systemic nAChR agonist that interferes with signal transduction in the central nervous system of sucking pests. Despite claims that FPF is potentially "bee-safe" by risk assessments, laboratory data have suggested that FPF has multiple sub-lethal effects on individual honey bees. Our study aimed to expand the studies to the effects of field-realistic concentration of FPF. We found a statistically significant decrease in the survival rate of honey bees exposed to FPF, whereas there were no significantly negative effects on larvae development durations nor foraging activity. In addition, we found that the exposed foragers showed significantly higher expression of ApidNT, CYP9Q2, CYP9Q3, and AmInR-2 compared to the CK group (control group), but no alteration in the gene expression was observed in larvae. The exposed newly emerged bees showed significantly higher expression of Defensin and ApidNT. These results indicate that the chronic exposure to the field-realistic concentration of FPF has negligible effects, but more important synergistic and behavioral effects that can affect colony fitness should be explored in the future, considering the wide use of FPF on crops pollinated and visited by honey bees.

The Larvicidal and Adulticidal Effects of Selected Plant Essential Oil Constituents on Greater Wax Moths.

Some plant essential oil constituents, such as monoterpenoids and phenylpropanoids, are promising insecticides in some situations and for certain insect pests. They vary in their toxicity, depending on the target insect. Moths (Lepidoptera) appear susceptible to these compounds, making them of promise for use against greater wax moths (Galleria mellonella Fabricius, (Lepidoptera: Pyrallidae), GWM), an important pest of western honey bee (Apis mellifera Linnaeus. (Hymenoptera: Apidae)) colonies. We determined the LC50 for GWM larvae or LD50 for GWM adults of select compounds (thymol, carvacrol, (S)-(+)-carvone, estragole, citral, linalool, (S)-(-)-limonene, and γ-terpinene). Concentrations between 8 and 2,266 µg/cm3 were mixed into the diets of GWM larvae and doses ranging between 0.08 and 70.3 mg per adult were applied topically to the abdomens of GWM adults. Lethal concentrations and doses were calculated after of 72 h of exposure. All eight compounds showed insecticidal activity against all tested stages of GWMs. Thymol (LC50 µg/cm3 (95% CI) = 21 [9-56], carvacrol = 46 [26-79], citral = 63 [30-134], and carvone = 76 [33-201]) had the highest toxicity toward GWM larvae. The hydrocarbons limonene (296 [231-377]), estragole (466 [354-611]), and γ-terpinene (729 [630-857]) had the lowest toxicity toward GWM larvae. Thymol had the highest toxicity towards GWM adults (LD50 (CI) = 0.5 [0.4-0.8] mg/adult). However, carvacrol (11.6 [10.1-13.6]), linalool (12.9 [9.3-17.8]), and limonene (15.8 [13.1-19.2]) were less toxic to GWM larvae. Our data show that select plant essential oil constituents are promising controls of GWM.

Effects of a commercially formulated glyphosate solutions at recommended concentrations on honeybee (Apis mellifera L.) behaviours.

Glyphosate, the active ingredient of the most widely used commercial herbicide formulation, is extensively used and produced in China. Previous studies have reported sublethal effects of glyphosate on honeybees. However, the effects of commercially formulated glyphosate (CFG) at the recommended concentration (RC) on the chronic toxicity of honeybees, especially on their behaviours, remain unknown. In this study, a series of behavioural experiments were conducted to investigate the effects of CFG on honeybees. The results showed that there was a significant decline in water responsiveness at 1/2 × , 1 × and 2 × the RC after 3 h of exposure to CFG for 11 days. The CFG significantly reduced sucrose responsiveness at 1/2 × and 1 × the RC. In addition, CFG significantly affected olfactory learning ability at 1/2 × , 1 × , and 2 × the RC and negatively affected memory ability at 1/2 × and 1 × the RC. The climbing ability of honeybees also significantly decreased at 1/2 × , 1 × and 2 × the RC. Our findings indicated that, after they were chronically exposed to CFG at the RC, honeybees exhibited behavioural changes. These results provide a theoretical basis for regulating field applications of CFG, which is necessary for establishing an early warning and notification system and for protecting honeybees.