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.

Flupyradifurone negatively affects survival, physical condition and mobility in the two-spotted lady beetle (Adalia bipunctata).

Lady beetles play a crucial role in natural ecosystems and agricultural settings. Unfortunately, these insects and more specifically the two-spotted lady beetle (Adalia bipunctata) are currently facing a severe decline in populations due to various stressors, with pesticide exposure being a significant threat. Flupyradifurone is a relatively newly introduced insecticide and as existing research is mainly elucidating its effects on bees there remains a limited understanding of its effects on non-hymenopteran insects, including lady beetles. In this study we investigated the impact of acute orally applied flupyradifurone doses on survival and sublethal parameters such as physical condition and mobility on A. bipunctata. Our findings revealed a significant increase in mortality among individuals subjected to flupyradifurone doses of 19 ng/individual (corresponding to >1.5-2.0 ng active substance (a.s.)/mg body weight (bw). The calculated LD50 of flupyradifurone at 48 h was 2.11 ng a.s./mg bw corresponding to an amount of 26.38 ng/individual. Sublethal consequences were observable immediately after pesticide application. Even at doses as low as 2 ng/individual (corresponding to >0.0-0.5 ng a.s./mg bw), flupyradifurone induced trembling and temporary immobility in treated animals. Furthermore, pesticide intoxication led to hypoactivity, with less distance covered and a decline in straightness of locomotion. In conclusion, our study underscores the harmful effects of flupyradifurone on the two-spotted lady beetle at doses notably lower than those affecting bees. These findings stress the importance of additional research to attain a more holistic understanding of pesticide impacts not only on a broader range of non-target arthropods species, but also on various exposure routes as well as lethal and sublethal effects.

Gamma-hydroxybutyrate (GHB), 1,4-butanediol (1,4BD), and gamma-butyrolactone (GBL) intoxication: A state-of-the-art review.

γ-hydroxybutyrate (GHB) is synthesized endogenously from γ-aminobutyric acid (GABA) or exogenously from 1,4-butanediol (butane-1,4-diol; 1,4-BD) or γ-butyrolactone (GBL). GBL, and 1,4-BD are rapidly converted to GHB. The gastric absorption time, volume of distribution, and half-life of GHB are between 5 and 45 min, 0.49 ± 0.9 L/kg, and between 20 and 60 min, respectively. GHB and its analogues have a dose-dependent effect on the activation of GHB receptor, GABA-B, and GABA localized to the central nervous system. After ingestion, most patients present transient neurological disorders (lethal dose: 60 mg/kg). Chronic GHB consumption is associated with disorders of use and a withdrawal syndrome when the consumption is discontinued. GHB, GBL, and 1,4-BD are classified as narcotics but only the use of GHB is controlled internationally. They are used for drug facilitated (sexual) assault, recreational purposes, slamsex, and chemsex. To confirm an exogenous intake or administration of GHB, GBL, or 1-4-BD, the pre-analytical conservation is crucial. The antemortem cutoff doses for detection are 5 and 5-15 mg/L, with detection windows of 6 and 10 h in the blood and urine, respectively Control of GHB is essential to limit the number of users, abuse, associated risks, and death related to their consumption.

N-3-oxododecanoyl homoserine lactone exacerbates endothelial cell death by inducing receptor-interacting protein kinase 1-dependent apoptosis.

Endothelial dysfunction is associated with the initiation of sepsis-associated organ failure. Bacterial quorum-sensing molecules act as pathogen-associated molecular patterns; however, the effects of quorum-sensing molecules on endothelial cells remain less understood. This study investigated the molecular mechanisms of quorum-sensing molecule-induced cell death and their interaction with lipopolysaccharide (LPS) in human umbilical vein endothelial cells. Endothelial cells were treated with N-3-oxododecanoyl homoserine lactone (3OC12-HSL) and LPS derived from Pseudomonas aeruginosa. Treatment with 3OC12-HSL reduced cell viability in a dose-dependent manner, and cotreatment with 3OC12-HSL and LPS enhanced cell death. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay revealed an increase in apoptotic cell death following 3OC12-HSL treatment; furthermore, cotreatment with 3OC12-HSL and LPS enhanced apoptosis. Western blotting revealed that treatment with 3OC12-HSL activated the receptor-interacting protein kinase 1 (RIPK1) pathway, leading to an increase in the levels of cleaved caspase 8 and 3. In addition, we found that treatment with necrostatin-1, an RIPK1 inhibitor, reduced cell death and ameliorated the activation of the RIPK1-dependent apoptotic pathway in 3OC12-HSL-treated cells. In conclusion, 3OC12-HSL induced endothelial cell apoptosis via the activation of the RIPK1 pathway, independent of LPS toxicity. Inhibition of RIPK1 may act as a therapeutic option for preserving endothelial cell integrity in patients with sepsis by disrupting the mechanism by which quorum-sensing molecules mediate their toxicity.

Long-term field-realistic exposure to a next-generation pesticide, flupyradifurone, impairs honey bee behaviour and survival.

The assessment of pesticide risks to insect pollinators have typically focused on short-term, lethal impacts. The environmental ramifications of many of the world's most commonly employed pesticides, such as those exhibiting systemic properties that can result in long-lasting exposure to insects, may thus be severely underestimated. Here, seven laboratories from Europe and North America performed a standardised experiment (a ring-test) to study the long-term lethal and sublethal impacts of the relatively recently approved 'bee safe' butenolide pesticide flupyradifurone (FPF, active ingredient in Sivanto®) on honey bees. The emerging contaminant, FPF, impaired bee survival and behaviour at field-realistic doses (down to 11 ng/bee/day, corresponding to 400 µg/kg) that were up to 101-fold lower than those reported by risk assessments (1110 ng/bee/day), despite an absence of time-reinforced toxicity. Our findings raise concerns about the chronic impact of pesticides on pollinators at a global scale and support a novel methodology for a refined risk assessment.

A toxicogenomics approach reveals characteristics supporting the honey bee (Apis mellifera L.) safety profile of the butenolide insecticide flupyradifurone.

Flupyradifurone, a novel butenolide insecticide, selectively targets insect nicotinic acetylcholine receptors (nAChRs), comparable to structurally different insecticidal chemotypes such as neonicotinoids and sulfoximines. However, flupyradifurone was shown in acute toxicity tests to be several orders of magnitude less toxic to western honey bee (Apis mellifera L.) than many other insecticides targeting insect nAChRs. The underlying reasons for this difference in toxicity remains unknown and were investigated here. Pharmacokinetic studies after contact application of [14C]flupyradifurone to honey bees revealed slow uptake, with internalized compound degraded into a few metabolites that are all practically non-toxic to honey bees in both oral and contact bioassays. Furthermore, receptor binding studies revealed a lack of high-affinity binding of these metabolites to honey bee nAChRs. Screening of a library of 27 heterologously expressed honey bee cytochrome P450 enzymes (P450s) identified three P450s involved in the detoxification of flupyradifurone: CYP6AQ1, CYP9Q2 and CYP9Q3. Transgenic Drosophila lines ectopically expressing CYP9Q2 and CYP9Q3 were significantly less susceptible to flupyradifurone when compared to control flies, confirming the importance of these P450s for flupyradifurone metabolism in honey bees. Biochemical assays using the fluorescent probe substrate 7-benzyloxymethoxy-4-(trifluoromethyl)-coumarin (BOMFC) indicated a weak, non-competitive inhibition of BOMFC metabolism by flupyradifurone. In contrast, the azole fungicides prochloraz and propiconazole were strong nanomolar inhibitors of these flupyradifurone metabolizing P450s, explaining their highly synergistic effects in combination with flupyradifurone as demonstrated in acute laboratory contact toxicity tests of adult bees. Interestingly, the azole fungicide prothioconazole is only slightly synergistic in combination with flupyradifurone - an observation supported by molecular P450 inhibition assays. Such molecular assays have value in the prediction of potential risks posed to bees by flupyradifurone mixture partners under applied conditions. Quantitative PCR confirmed the expression of the identified P450 genes in all honey bee life-stages, with highest expression levels observed in late larvae and adults, suggesting honey bees have the capacity to metabolize flupyradifurone across all life-stages. These findings provide a biochemical explanation for the low intrinsic toxicity of flupyradifurone to honey bees and offer a new, more holistic approach to support bee pollinator risk assessment by molecular means.

Flupyradifurone reduces nectar consumption and foraging but does not alter honey bee recruitment dancing.

Foraging is essential for honey bee colony fitness and is enhanced by the waggle dance, a recruitment behavior in which bees can communicate food location and quality. We tested if the consumption of nectar (sucrose solution) with a field-realistic concentration of 4 ppm flupyradifurone (FPF) could alter foraging behavior and recruitment dancing in Apis mellifera. Foragers were repelled by FPF. They visited the FPF feeder less often and spent less time imbibing sucrose solution (2.5 M, 65% w/w) with FPF. As a result, bees feeding on the FPF treatment consumed 16% less nectar. However, FPF did not affect dancing: there were no effects on unloading wait time, the number of dance bouts per nest visit, or the number of dance circuits performed per dance bout. FPF could therefore deter bees from foraging on contaminated nectar. However, the willingness of bees to recruit nestmates for nectar with FPF is concerning. Recruitment can rapidly amplify the number of foragers and could overcome the decrease in consumption of FPF-contaminated nectar, resulting in a net inflow of pesticide to the colony. FPF also significantly altered the expression of 116 genes, some of which may be relevant for the olfactory learning deficits induced by FPF and the toxicity of FPF.

Diffusible Signaling Factor, a Quorum-Sensing Molecule, Interferes with and Is Toxic Towards Bdellovibrio bacteriovorus 109J.

Bdellovibrio bacteriovorus 109J is a predatory bacterium which lives by predating on other Gram-negative bacteria to obtain the nutrients it needs for replication and survival. Here, we evaluated the effects two classes of bacterial signaling molecules (acyl homoserine lactones (AHLs) and diffusible signaling factor (DSF)) have on B. bacteriovorus 109J behavior and viability. While AHLs had a non-significant impact on predation rates, DSF considerably delayed predation and bdelloplast lysis. Subsequent experiments showed that 50 µM DSF also reduced the motility of attack-phase B. bacteriovorus 109J cells by 50% (38.2 ± 14.9 vs. 17 ± 8.9 µm/s). Transcriptomic analyses found that DSF caused genome-wide changes in B. bacteriovorus 109J gene expression patterns during both the attack and intraperiplasmic phases, including the significant downregulation of the flagellum assembly genes and numerous serine protease genes. While the former accounts for the reduced speeds observed, the latter was confirmed experimentally with 50 µM DSF completely blocking protease secretion from attack-phase cells. Additional experiments found that 30% of the total cellular ATP was released into the supernatant when B. bacteriovorus 109J was exposed to 200 µM DSF, implying that this QS molecule negatively impacts membrane integrity.

Evaluation of potential miticide toxicity to Varroa destructor and honey bees, Apis mellifera, under laboratory conditions.

The honey bee, Apis mellifera L., is the world's most important managed pollinator of agricultural crops, however, Varroa mite, Varroa destructor Anderson and Trueman, infestation has threatened honey bee survivorship. Low efficacy and development of Varroa mite resistance to currently used Varroacides has increased the demand for innovative, effective treatment tool options that exhibit high efficacy, while minimizing adverse effects on honey bee fitness. In this investigation, the toxicity of 16 active ingredients and 9 formulated products of registered miticides for use on crops from 12 chemical families were evaluated in comparison to amitraz on Varroa mites and honey bees using contact surface and topical exposures. It was found that fenpyroximate (93% mortality), spirotetramat (84% mortality) and spirodiclofen (70% mortality) had greater toxicity to Varroa mites, but high dose rates caused high bee mortality (> 60%). With this in mind, further research is needed to investigate other options to minimize the adverse effect of these compounds on bees. The results also found high toxicity of fenazaquin and etoxazole against Varroa mites causing 92% and 69% mortality, respectively; and were found to be safe on honey bees. Collectively, it is recommended that fenazaquin and etoxazole are candidates for a potential Varroacide and recommended for further testing against Varroa mites at the colony level.

N-(3-oxododecanoyl)-homoserine lactone regulates osteoblast apoptosis and differentiation by mediating intracellular calcium.

Pseudomonas aeruginosa (P. aeruginosa) is associated with periapical periodontitis. The lesions are characterized by a disorder in osteoblast metabolism. Quorum sensing molecular N-(3-oxododecanoyl)-homoserine lactone (AHL) is secreted by P. aeruginosa and governs the expression of numerous virulence factors. AHL can trigger intracellular calcium ([Ca2+]i) fluctuations in many host cells. However, it is unclear whether AHL can regulate osteoblast metabolism by affecting [Ca2+]i changes or its spatial correlation. We explored AHL-induced apoptosis and differentiation in pre-osteoblastic MC3T3-E1 cells and evaluated [Ca2+]i mobilization using several extraction methods. The spatial distribution pattern of [Ca2+]i among cells was investigated by Moran's I, an index of spatial autocorrelation. We found that 30 µM and 50 µM AHL triggered opposing osteoblast fates. At 50 µM, AHL inhibited osteoblast differentiation by promoting mitochondrial-dependent apoptosis and negatively regulating osteogenic marker genes, including Runx2, Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). In contrast, prolonged treatment with 30 µM AHL promoted osteoblast differentiation concomitantly with cell apoptosis. The elevation of [Ca2+]i levels in osteoblasts treated with 50 µM AHL was spatially autocorrelated, while no such phenomenon was observed in 30 µM AHL-treated osteoblasts. The blocking of cell-to-cell spatial autocorrelation in the osteoblasts provoked by 50 µM AHL significantly inhibited apoptosis and partially restored differentiation. Our observations suggest that AHL affects the fate of osteoblasts (apoptosis and differentiation) by affecting the spatial correlation of [Ca2+]i changes. Thus, AHL acts as a double-edged sword for osteoblast function.

Sarcophine and (7S, 8R)-dihydroxydeepoxysarcophine from the Red Sea soft coral Sarcophyton glaucum as in vitro and in vivo modulators of glycine receptors.

The inhibitory glycine receptor (GlyR) is a key mediator of synaptic signalling in spinal cord, brain stem, and higher centres of the central nervous system. We examined the glycinergic activity of sarcophine (SN), a marine terpenoid known for its various biological activities, and its trans-diol derivative (7S, 8R)-dihydroxy-deepoxysarcophine (DSN). SN was isolated from the Red Sea soft coral Sacrophyton glaucum, DSN was semisynthesized by hydrolysis of the epoxide ring. In cytotoxicity tests against HEK293 cells, SN and DSN had LD50 values of 29.3 ± 3.0 mM and 123.5 ± 13.0 mM, respectively. Both compounds were tested against recombinant human α1 glycine receptors in HEK293 cells using whole-cell recording techniques. Both, SN and DSN were shown for the first time to be inhibitors of recombinant glycine receptors, with KIvalues of 2.1 ± 0.3 µM for SN, and 109 ± 9 µM for DSN. Receptor inhibition was also studied in vivo in a mouse model of strychnine toxicity. Surprisingly, in mouse experiments strychnine inhibition was not augmented by either terpenoid. While DSN had no significant effect on strychnine toxicity, SN even delayed strychnine effects. This could be accounted for by assuming that strychnine and sarcophine derivatives compete for the same binding site on the receptor, so the less toxic sarcophine can prevent strychnine from binding. The combination of modulatory activity and low level of toxicity makes sarcophines attractive structures for novel glycinergic drugs.

Synthesis, molecular docking studies, and larvicidal activity evaluation of new fluorinated neonicotinoids against Anopheles darlingi larvae.

Anopheles darlingi is the main vector of malaria in Brazil, characterized by a high level of anthropophilia and endophagy. Imidacloprid, thiacloprid, and acetamiprid are the most widespread insecticides of the neonicotinoid group. However, they produce adverse effects on the non-target insects. Flupyradifurone has been marketed as an alternative to non-fluorinated neonicotinoids. Neonicotinoids containing trifluoroacethyl substituent reveal increased insecticidal activity due to higher hydrophobicity and metabolic stability. We synthesized novel neonicotinoid insecticides containing fluorinated acceptor groups and their interactions were estimated with the nicotinic acetylcholine receptor (nAChR) binding site by molecular docking studies, to evaluate their larvicidal activity against A. darlingi, and to assess their outdoor photodegradation behavior. New neonicotinoid analogues were prepared and characterized by NMR and mass-spectrometry. The synthesized molecules were modelled by time-dependent density functional theory and analyzed, their interaction with nAChR was investigated by molecular docking. Their insecticide activity was tested on Anopheles larvae collected in suburban area of Manaus, Brazil. Four new fluorinated neonicotinoid analogs were prepared and tested against 3rd instars larvae of A. darlingi showing high larvicidal activity. Docking studies reveal binding modes of the synthesized compounds and suggest that their insecticidal potency is governed by specific interactions with the receptor binding site and enhanced lipophilicity. 2-Chloro-5-(2-trifluoromethyl-pyrrolidin-1-ylmethyl)pyridine 5 showed fast degradation in water maintaining high larvicidal activity. All obtained substances possessed high larvicidal activity in low concentrations in 48 hours of exposure, compared to commercial flupyradifurone. Such activity is connected to a unique binding pattern of the synthesized compounds to insect's nAChR and to their enhanced bioavailability owing to introduction of fluorinated amino-moieties. Therefore, the compounds in question have a high potential for application as control agents for insects transmitting tropical diseases, and they will be less persistent in the environment.

Consequences of a short time exposure to a sublethal dose of Flupyradifurone (Sivanto) pesticide early in life on survival and immunity in the honeybee (Apis mellifera).

Dramatic losses of pollinating insects have become of global concern, as they threaten not only key ecosystem services but also human food production. Recent research provided evidence that interactions between ecological stressors are drivers of declining pollinator health and responsible for observed population collapses. We used the honeybee Apis mellifera and conducted a series of experiments to test for long-term effects of a single short exposure to the agricultural pesticide flupyradifurone to a second environmental stressor later in life. To do this, we exposed individuals during their larval development or early adulthood to sublethal dosages of flupyradifurone (0.025 µg for larvae and 0.645 µg for imagos), either pure or as part of an agricultural formulation (Sivanto). We afterwards exposed bees to a second ecological stressor infecting individuals with 10,000 spores of the fungal gut parasite Nosema ceranae. We found that pesticide exposures significantly reduced survival of bees and altered the expression of several immune and detoxification genes. The ability of bees to respond to these latter effects differed significantly between colonies, offering opportunities to breed bees with elevated levels of pesticide tolerance in the future. We conclude that short episodes of sublethal pesticide exposures during development are sufficient to trigger effects later in life and could therefore contribute to the widespread declines in bee health.