Investigating the thermal sensitivity of key enzymes involved in the energetic metabolism of three insect species.

The metabolic responses of insects to high temperatures have been linked to their mitochondrial substrate oxidation capacity. However, the mechanism behind this mitochondrial flexibility is not well understood. Here, we used three insect species with different thermal tolerances (the honey bee, Apis mellifera; the fruit fly, Drosophila melanogaster; and the potato beetle, Leptinotarsa decemlineata) to characterize the thermal sensitivity of different metabolic enzymes. Specifically, we measured activity of enzymes involved in glycolysis (hexokinase, HK; pyruvate kinase, PK; and lactate dehydrogenase, LDH), pyruvate oxidation and the tricarboxylic acid cycle (pyruvate dehydrogenase, PDH; citrate synthase, CS; malate dehydrogenase, MDH; and aspartate aminotransferase, AAT), and the electron transport system (Complex I, CI; Complex II, CII; mitochondrial glycerol-3-phosphate dehydrogenase, mG3PDH; proline dehydrogenase, ProDH; and Complex IV, CIV), as well as that of ATP synthase (CV) at 18, 24, 30, 36, 42 and 45°C. Our results show that at high temperature, all three species have significantly increased activity of enzymes linked to FADH2 oxidation, specifically CII and mG3PDH. In fruit flies and honey bees, this coincides with a significant decrease of PDH and CS activity, respectively, that would limit NADH production. This is in line with the switch from NADH-linked substrates to FADH2-linked substrates previously observed with mitochondrial oxygen consumption. Thus, we demonstrate that even though the three insect species have a different metabolic regulation, a similar response to high temperature involving CII and mG3PDH is observed, denoting the importance of these proteins for thermal tolerance in insects.

Phylogenomic and functional characterization of an evolutionary conserved cytochrome P450-based insecticide detoxification mechanism in bees.

The regulatory process for assessing the risks of pesticides to bees relies heavily on the use of the honeybee, Apis mellifera, as a model for other bee species. However, the validity of using A. mellifera as a surrogate for other Apis and non-Apis bees in pesticide risk assessment has been questioned. Related to this line of research, recent work on A. mellifera has shown that specific P450 enzymes belonging to the CYP9Q subfamily act as critically important determinants of insecticide sensitivity in this species by efficiently detoxifying certain insecticide chemotypes. However, the extent to which the presence of functional orthologs of these enzymes is conserved across the diversity of bees is unclear. Here we used a phylogenomic approach to identify > 100 putative CYP9Q functional orthologs across 75 bee species encompassing all major bee families. Functional analysis of 26 P450s from 20 representative bee species revealed that P450-mediated detoxification of certain systemic insecticides, including the neonicotinoid thiacloprid and the butenolide flupyradifurone, is conserved across all major bee pollinator families. However, our analyses also reveal that CYP9Q-related genes are not universal to all bee species, with some Megachilidae species lacking such genes. Thus, our results reveal an evolutionary conserved capacity to metabolize certain insecticides across all major bee families while identifying a small number of bee species where this function may have been lost. Furthermore, they illustrate the potential of a toxicogenomic approach to inform pesticide risk assessment for nonmanaged bee species by predicting the capability of bee pollinator species to break down synthetic insecticides.

The periplasmic expression and purification of AA15 lytic polysaccharide monooxygenases from insect species in Escherichia coli.

Lytic polysaccharide monooxygenases (LPMOs) are metalloenzymes that cleave structural polysaccharides through an oxidative mechanism. The enzymatic activity of LPMOs relies on the presence of a Cu2+ histidine-brace motif in their flat catalytic surface. Upon reduction by an external electron donor and in the presence of its co-substrates, O2 or H2O2, LPMOs can generate reactive oxygen species to oxidize the substrates. Fungal and bacterial LPMOs are involved in the catabolism of polysaccharides, such as chitin, cellulose, and hemicelluloses, and virulence mechanisms. Based on the reports on the discovery of LPMOs from the family AA15 in termites, firebrats, and flies, the functional role of the LPMO in the biosphere could expand, as these enzymes may be correlated with chitin remodeling and molting in insects. However, there is limited knowledge of AA15 LPMOs due to difficulties in recombinant expression of soluble proteins and purification protocols. In this study, we describe a protocol for the cloning, expression, and purification of insect AA15 LPMOs from Arthropoda, mainly from termites, followed by the expression and purification of an AA15 LPMO from the silkworm Bombyx mori, which contains a relatively high number of disulfide bonds. We also report the recombinant expression and purification of a protein with homology to AA15 family from the western European honeybee Apis mellifera, an LPMO-like enzyme lacking the canonical histidine brace. Therefore, this work can support future studies concerning the role of LPMOs in the biology of insects and inspire molecular entomologists and insect biochemists in conducting activities in this field.

Telomerase-Positive Somatic Tissues of Honeybee Queens (Apis mellifera) Display No DNA Replication.

Telomere biology is closely linked to the process of aging. The restoration of telomere length by maintaining telome-rase activity in certain cell types of human adults allows for the proliferative capacity of the cells and preserves the regeneration potential of the tissue. The absence of telome-rase, that leads to telomere attrition and irreversible cell cycle arrest in most somatic cells, acts as a protective mechanism against uncontrolled cancer growth. Nevertheless, there have been numerous studies indicating noncanonical functions of telomerase besides those involved in telomere lengthening. Eusocial insects serve as a great system for aging research. This is because eusocial reproductives, such as queens and kings, have a significantly extended lifespan compared to nonreproductive individuals of the same species. We report that the somatic tissues of honeybee queens (Apis mellifera) are associated with upregulated telomerase activity; however, this upregulation does not fully correlate with the rate of DNA replication in the tissues. This indicates a noncanonical role of telomerase in the somatic tissues of honeybee queens.

Floral origin modulates the content of a lipid marker in Apis mellifera honey.

The compound DAGE (DiAcyl Glyceryl Ether, 1-stearyl-2,3-dioleoyl glycerol), present in Apis mellifera honey, is a lipidic entomological marker secreted by the salivary glands of worker bees. Its content was determined by NMR, analyzing the organic extracts of a number of Italian honeys of different floral typology. We have found that the DAGE content is related to the botanical origin of honey. This dependence on floral typology was further confirmed by a linear correlation (R2 > 0.83) observed between the content of DAGE and the enzymatic activity of invertase and diastase in honey. Also these enzymes originate from bee salivary secretions and their concentrations in honey are known to depend on the floral source. DAGE content appears to be a sensitive parameter to some forms of honey manipulations, as indicated by the results of artificial bee-feeding experiments. This suggests its possible use as indicator of honey authenticity.

Lipolytic Activity of a Carboxylesterase from Bumblebee (Bombus ignitus) Venom.

Bee venom is a complex mixture composed of peptides, proteins with enzymatic properties, and low-molecular-weight compounds. Although the carboxylesterase in bee venom has been identified as an allergen, the enzyme's role as a venom component has not been previously elucidated. Here, we show the lipolytic activity of a bumblebee (Bombus ignitus) venom carboxylesterase (BivCaE). The presence of BivCaE in the venom secreted by B. ignitus worker bees was confirmed using an anti-BivCaE antibody raised against a recombinant BivCaE protein produced in baculovirus-infected insect cells. The enzymatic activity of the recombinant BivCaE protein was optimal at 40 °C and pH 8.5. Recombinant BivCaE protein degrades triglycerides and exhibits high lipolytic activity toward long-chain triglycerides, defining the role of BivCaE as a lipolytic agent. Bee venom phospholipase A2 binds to mammalian cells and induces apoptosis, whereas BivCaE does not affect mammalian cells. Collectively, our data demonstrate that BivCaE functions as a lipolytic agent in bee venom, suggesting that BivCaE will be involved in distributing the venom via degradation of blood triglycerides.

Understanding the binding mechanism of succinic acid against phospholipase A2 from bee venom.

Phospholipase A2 (PLA2 ) is responsible for the release of fatty acids from glycerophospholipids. PLA2 is commonly found in mammalian tissues. It is also found in venom from different animals ranging from insects, arachnid, and snakes. The release of arachidonic acid in large amount results in inflammation and pain. Identification of compounds that can inhibit the activity of PLA2 is of large scientific and medicinal interest as these compounds can act as antidotes toward snake bites and bee stings. Among the different compounds that have been tested for inhibition of PLA2 , a secondary metabolite succinic acid is identified to inhibit PLA2 activity. The inhibition was analyzed using an in vitro PLA2 inhibition assay and isothermal titration calorimetry (ITC) studies. The molecular mechanism of the mode of inhibition was studied using molecular docking and simulation studies.

Phospholipase A2 way to hydrolysis: Dint formation, hydrophobic mismatch, and lipid exclusion.

Phospholipase A2 (PLA2) exerts a wide range of biological effects and attracts a lot of attention of researchers. Two sites are involved in manifestation of PLA2 enzymatic activity: catalytic site responsible for substrate binding and fatty acid cleavage from the sn-2 position of a glycerophospholipid, and interface binding site (IBS) responsible for the protein binding to lipid membrane. IBS is formed by positively charged and hydrophobic amino acids on the outer surface of the protein molecule. Understanding the mechanism of PLA2 interaction with the lipid membrane is the most challenging step in biochemistry of this enzyme. We used a combination of experimental and computer simulation techniques to clarify molecular details of bee venom PLA2 interaction with lipid bilayers formed by palmitoyloleoylphosphatidylcholine or dipalmitoylphosphatidylcholine. We found that after initial enzyme contact with the membrane, a network of hydrogen bonds was formed. This led to deformation of the interacting leaflet and dint formation. The bilayer response to the deformation depended on its phase state. In a gel-phase bilayer, diffusion of lipids is restricted therefore chain melting occurred in both leaflets of the bilayer. In the case of a fluid-phase bilayer, lateral diffusion is possible, and lipid polar head groups were excluded from the contact area. As a result, the bilayer became thinner and a large hydrophobic area was formed. We assume that relative ability of a bilayer to come through lipid redistribution process defines the rate of initial stages of the catalysis.

Differences in larval pesticide tolerance and esterase activity across honey bee (Apis mellifera) stocks.

Honey bee populations in North America are an amalgamation of diverse progenitor ecotypes experiencing varying levels of artificial selection. Genetic differences between populations can result in variable susceptibility towards environmental stressors, and here we compared pesticide tolerances across breeding stocks using a mixture of seven pesticides frequently found in colonies providing pollination services. We administered the pesticide mixture chronically to in vitro reared larvae at four concentrations of increasing Hazard Quotient (HQ, or cumulative toxicity) and measured mortality during larval development. We found that different stocks had significantly different tolerances to our pesticide mixture as indicated by their median lethal toxicity (HQ50). The intensively selected Pol-Line stock exhibited the greatest pesticide sensitivity while Old World (progenitor) and putatively feral stocks were the most pesticide-tolerant. Furthermore, we found that activity of the detoxification enzyme esterase was positively correlated with pesticide tolerance when measured using two different substrate standards, and confirmed that larvae from the Pol-Line stock had generally lower esterase activity. Consistent with an increased pesticide tolerance, the Old World and putatively feral stocks had higher esterase activities. However, esterases and other detoxification enzymes (CYP450s and GSTs) were found in similar abundances across stocks, suggesting that the differences in enzyme activity we observed might arise from stock-specific single nucleotide polymorphisms or post-translational modifications causing qualitative variation in enzyme activity. These results suggest that selective breeding may inadvertently increase honey bees' sensitivity to pesticides, whereas unselected, putatively feral and Old World stocks have larvae that are more tolerant.

Effect of age on insecticide susceptibility and enzymatic activities of three detoxification enzymes and one invertase in honey bee workers (Apis mellifera).

Honey bee is an economically important insect for honey production and pollination. Frequent exposure to toxic pesticides is one of the major risk factors causing the pollinator population decline. However, age effects of honey bees on pesticide susceptibility have been largely ignored and many researchers use bees of unknown age for assessing the risk of pesticides. Honey bee workers are known to go through physiological and behavioral changes in order to differentiate different phenotypes to perform specific duties over their natural lifetime of 6 weeks or longer. In this study, we provide multi-parameter evidences of unignorable age effects of honey bee workers and suggest using a standard bee age to produce reliable and comparable data when assessing variable and realistic situations of in-hive and field exposures to pesticides. Using honey bee workers aged 4- to 42-days old, we examined susceptibility of the bees to five different insecticides from five different classes and measured enzymatic activities of three major detoxification enzymes and an invertase involved in honey production. Results showed gradual increase of natural mortality and decrease of soluble protein content in bees over the age span from 4 days to 42 days. Significant increases of mortality after separate treatments of five different insecticides confirmed drastic age effects of bees over the assessed age span. As they aged, honey bees also showed a gradual increase of cytochrome P450 oxidase activity while still maintaining constant levels of two other detoxification enzymes (esterase and glutathione S-transferase) and an invertase responsible for honey production.

Role of Apis cerana cerana N-terminal asparagine amidohydrolase (AccNtan1) in oxidative stress.

N-Terminal asparagine amidohydrolase is a component of the ubiquitin-dependent N-end rule pathway of protein degradation that has been implicated in a variety of physiological functions, including the sensing of heme, oxygen, nitric oxide, selective elimination of misfolded proteins and the repair of DNA. We identified the Apis cerana cerana N-terminal asparagine amidohydrolase (AccNtan1) gene from A. cerana cerana and investigated its role in oxidation resistance. Multiple sequence alignments and phylogenetic analysis revealed that N-terminal asparagine amidohydrolase is highly conserved in insect species. Quantitative real-time polymerase chain reaction analysis indicated that the expression levels of AccNtan1 were significantly lower in the wing, honey sac and abdomen than in other tissues and were significantly higher in early stages of development, including the larva, prepupa and pink-eyed pupa stages, than in later stages. We further observed that AccNtan1 expression was induced by several environmental stressors, including aberrant temperature, H2O2, UV, heavy metals and pesticides. Moreover, a bacteriostatic assay suggested that overexpression of AccNtan1 enhances the resistance of bacteria to oxidative stress. In addition, knockdown of AccNtan1 using RNA interference significantly affected the expression levels of most antioxidant genes and the activity levels of several antioxidant enzymes. Thus, we hypothesize that AccNtan1 plays important roles in environmental stress responses and antioxidative processes.

The IRE1 pathway regulates honey bee Unfolded Protein Response gene expression.

Our molecular understanding of honey bee cellular stress responses is incomplete. Previously, we sought to identify and began functional characterization of the components of the Unfolded Protein Response (UPR) in honey bees. We observed that UPR stimulation resulted in induction of target genes upon IRE1 pathway activation, as assessed by splicing of Xbp1 mRNA. However, we were not able to determine the relative role of the various UPR pathways in gene activation. Our understanding of honey bee signal transduction and transcriptional regulation has been hampered by a lack of tools. After using RNA-seq to expand the known UPR targets in the honey bee, we used the Drosophila melanogaster S2 cell line and honey bee trans and cis elements to investigate the role of the IRE1 pathway in the transcriptional activation of one of these targets, the honey bee Hsc70-3 gene. Using a luciferase reporter, we show that honey bee Hsc70 promoter activity is inducible by UPR activation. In addition, we show that this activation is IRE1-dependent and relies on specific cis regulatory elements. Experiments using exogenous honey bee or fruit fly XBP1S proteins demonstrate that both factors can activate the Hsc70-3 promoter and further support a role for the IRE1 pathway in control of Hsc70-3 expression in the honey bee. By providing foundational knowledge about the UPR in the honey bee and demonstrating the usefulness of a heterologous cell line for molecular characterization of honey bee pathways, this work stands to improve our understanding of this critical species.

Molecular dynamics reveals insight into how N226P and H227Y mutations affect maltose binding in the active site of α-glucosidase II from European honeybee, Apis mellifera.

European honeybee, Apis mellifera, produces α-glucosidase (HBGase) that catalyzes the cleavage of an α-glycosidic bond of the non-reducing end of polysaccharides and has potential applications for malt hydrolysis in brewing industry. Characterized by their substrate specificities, HBGases have three isoforms including HBGase II, which prefers maltose to sucrose as a substrate. Previous study found that the catalytic efficiency of maltose hydrolysis of N226P mutant of HBGase II was higher than that of the wild type (WT), and the catalytic efficiency of maltose hydrolysis of WT was higher than those of H227Y and N226P-H227Y mutants. We hypothesized that N226P mutation probably caused maltose to bind with better affinity and position/orientation for hydrolysis than WT, while H227Y and N226P-H227Y mutations caused maltose to bind with worse affinity and position/orientation for hydrolysis than WT. Using this hypothesis, we performed molecular dynamics on the catalytically competent binding conformations of maltose/WT, maltose/N226P, maltose/H227Y, and maltose/N226P-H227Y complexes to elucidate effects of N226P and H227Y mutations on maltose binding in HBGase II active site. Our results reasonably support this hypothesis because the N226P mutant had better binding affinity, higher number of important binding residues, strong and medium hydrogen bonds as well as shorter distance between atoms necessary for hydrolysis than WT, while the H227Y and N226P-H227Y mutants had worse binding affinities, lower number of important binding residues and strong hydrogen bonds as well as longer distances between atoms necessary for hydrolysis than WT. Moreover, results of binding free energy and hydrogen bond interaction of residue 227 support the role of H227 as a maltose preference residue, as proposed by previous studies. Our study provides important and novel insight into how N226P and H227Y mutations affect maltose binding in HBGase II active site. This knowledge could potentially be used to engineer HBGase II to improve its efficiency.

Illustrating Interfacial Interaction between Honey Bee Venom Phospholipase A2 and Supported Negatively Charged Lipids with Sum Frequency Generation and Laser Scanning Confocal Microscopy.

Phospholipase A2 is an important enzyme species which can widely be found in animals, plants, bacteria, and so on. A large number of studies have shown that phospholipase A2 is highly catalytic toward the lipids. Here, sum frequency generation (SFG) vibrational spectroscopy and laser scanning confocal microscopy (LSCM) were applied to study the interaction between honey bee venom phospholipase A2 (bvPLA2) and the negatively charged DPPG bilayer. In both cases without and with the calcium ions (Ca2+), the bvPLA2 molecules were adsorbed onto the outer leaflet surface with the orientational order, and the adsorbed bvPLA2 molecules damaged the order of the packed outer leaflet. In comparison to the case without Ca2+, the addition of Ca2+ can accelerate the attaching process of bvPLA2 to the outer leaflet surface and decelerate the process of damaging the outer leaflet order. The experimental result also confirmed, with the help of the Ca2+, the DPPG molecules in the outer leaflet were hydrolyzed, with both hydrolysates, that is, lysophospholipids and fatty acids, remaining at the interface, showing a distinct difference from previous published literatures regarding neutral lipids [Phys. Chem. Chem. Phys. 2018, 20, 63-67] and PLA1 [Langmuir 2019, 35, 12831-12838].

Acute oral and contact toxicity of new ethyl-carbamates on the mortality and acetylcholinesterase activity of honey bee (Apis mellifera).

The effects produced by the ethyl-carbamates: ethyl-4-bromophenyl carbamate (LQM 919) and ethyl-4-chlorophenyl carbamate (LQM 996) on the mortality and behavior of Apis mellifera were evaluated by the acute oral toxicity test and the acute contact toxicity test. The oral lethal dose, 50% of the ethyl-carbamates was >145.24 µg per bee, and the oral lethal dose, 50% of propoxur was 0.072 µg per bee. Therefore, according to the OECD criteria, the ethyl-carbamates were classified as relatively nontoxic orally; meanwhile, propoxur was classified as highly toxic orally. In the contact test, lethal concentrations 50% of the ethyl-carbamates were 4.83 and 2.23 µg/cm2 for LQM 919 and LQM 996, respectively; therefore, they were at least 10-fold less lethal (p < 0.05) than propoxur (0.22 µg/cm2). The ethyl-carbamates reduced the activity of A. mellifera acetylcholinesterase by up to 30%. The ki and kd values of both ethyl-carbamates were lower (p < 0.05) than those of propoxur and indicated that they are weak inhibitors and with low affinity to A. mellifera acetylcholinesterase, which along with the absence of behavioral alterations suggests that the mortality caused by ethyl carbamates is not related to damage to the nervous system. According to these results, the evaluated ethyl-carbamates can be considered a low ecotoxic risk for A. mellifera.

The anticoagulant effect of Apis mellifera phospholipase A2 is inhibited by CORM-2 via a carbon monoxide-independent mechanism.

Bee venom phospholipase A2 (PLA2) has potential for significant morbidity. Ruthenium (Ru)-based carbon monoxide releasing molecules (CORM) inhibit snake venoms that are anticoagulant and contain PLA2. In addition to modulating heme-bearing proteins with carbon monoxide, these CORM generate reactive Ru species that form adducts with histamine residues resulting in changes in protein function. This study sought to identify anticoagulant properties of bee venom PLA2 via catalysis of plasma phospholipids required for thrombin generation. Another goal was to determine if Ru-based CORM inhibit bee venom PLA2 via carbon monoxide release or via potential binding of reactive Ru species to a key histidine residue in the catalytic site of the enzyme. Anticoagulant activity of bee venom PLA2 was assessed via thrombelastography with normal plasma. Bee venom PLA2 was then exposed to different CORM and a metheme forming agent and anticoagulant activity was reassessed. Using Ru, boron and manganese-based CORM and a metheme forming agent, it was demonstrated that it was unlikely that carbon monoxide interaction with a heme group attached to PLA2 was responsible for inhibition of anticoagulant activity by Ru-based CORM. Exposure of PLA2 to a Ru-based CORM in the presence of histidine-rich human albumin resulted in loss of inhibition of PLA2. Ru-based CORM likely inhibit bee venom PLA2 anticoagulant activity via formation of reactive Ru species that bind to histidine residues of the enzyme.

Telomerase activity is upregulated in the fat bodies of pre-diapause bumblebee queens (Bombus terrestris).

The attrition of telomeres, the ends of eukaryote chromosomes, and activity of telomerase, the enzyme that restores telomere length, play a role in the ageing process and act as indicators of biological age. A notable feature of advanced eusocial insects is the longevity of reproductive individuals (queens and kings) compared to those from non-reproductive castes (workers and soldiers) within a given species, with a proposed link towards upregulation of telomerase activity in the somatic tissues of reproductive individuals. Given this, eusocial insects provide excellent model systems for research into ageing. We tested telomerase activity and measured telomere length in Bombus terrestris, which is a primitively eusocial insect species with several distinct features compared to advanced social insects. In somatic tissues, telomerase activity was upregulated only in the fat bodies of pre-diapause queens, and this upregulation was linked to heightened DNA synthesis. Telomere length was shorter in old queens compared to that in younger queens or workers. We speculate that (1) the upregulation of telomerase activity, together with DNA synthesis, is the essential step for intensifying metabolic activity in the fat body to build up a sufficient energy reserve prior to diapause, and that (2) the lifespan differences between B. terrestris workers and queens are related to the long diapause period of the queen. A possible relationship between telomere length regulation and TOR, FOXO, and InR as cell signaling components, was tested.

The levels of natural Nosema spp. infection in Apis mellifera iberiensis brood stages.

Nosema ceranae is the most prevalent endoparasite of Apis mellifera iberiensis and it is a major health problem for bees worldwide. The infective capacity of N. ceranae has been demonstrated experimentally in honey bee brood, however no data are available about its prevalence in brood under natural conditions. Thus, brood combs from 10 different hives were analyzed over two consecutive years, taking samples before and after winter. A total of 1433 larvae/pupae were analyzed individually and N. ceranae (3.53%) was the microsporidian most frequently detected, as opposed to Nosema apis (0.42%) which was more frequently detected in conjunction with N. ceranae (0.71%). The active multiplication of both microsporidians was confirmed by the expression (real-time-PCR) of the N. ceranae polar tube protein 3 gene and/or the N. apis RNA polymerase II gene in 24% of the brood samples positive for Nosema spp. Both genes are related to microsporidian multiplication. As such, N. ceranae multiplication was confirmed in 1.06% of the samples, while N. apis multiplication was only observed in co-infections with N. ceranae (0.07%). Brood cells were analyzed for the presence of Nosema spp., as those are the immediate environment where the brood stages develop. The brood samples infected by Nosema spp. were in brood cells in which that microsporidians were not detected, while brood cells positive for N. ceranae hosted brood stages that were not apparently infected, indicating that this is unlikely to be the main pathway of infection. Finally, the colonies with brood infected by N. ceranae showed higher levels (numbers) of infected adult bees, although the differences were not significant before (P = 0.260), during (P = 0.055) or after (P = 0.056) brood sampling. These results show that N. ceranae is a bee parasite ubiquitous to all members of the colony, irrespective of the age of the bee. It is also of veterinary interest and should be considered when studying the epidemiology of the disease.

Toxicity of oxalic acid and impact on some antioxidant enzymes on in vitro-reared honeybee larvae.

Nowadays, Varroa destructor is considered as a serious pest of honeybees (Apis mellifera) and its resistance to acaricides has been reported in Europe since the early 1990s. That is why new methods of treatment for Varroa mites are still in focus of many scientists. In our study, we determined the lethal concentration LC50 (72 h) of 2.425% oxalic acid solution following single spray exposure of honeybee larvae under laboratory conditions (Guideline OECD 237 2013). Potential sublethal effects of oxalic acid were monitored through the determination of the activity of antioxidant enzymes. Activation of primary antioxidant enzymes was observed at 1.75% of oxalic acid; 3.5% of oxalic acid brought on a statistically significant increase of glutathione S-transferase activity. This change was accompanied by an increase in thiobarbituric acid reactive substances, products of lipid peroxidation. Our results indicate that oxalic acid may be harmful to bee brood when present during application.

Identification and functional characterisation of a novel N-cyanoamidine neonicotinoid metabolising cytochrome P450, CYP9Q6, from the buff-tailed bumblebee Bombus terrestris.

Recent work has shown that two bumblebee (Bombus terrestris) cytochrome P450s of the CYP9Q subfamily, CYP9Q4 and CYP9Q5, are important biochemical determinants of sensitivity to neonicotinoid insecticides. Here, we report the characterisation of a third P450 gene CYP9Q6, previously mis-annotated in the genome of B. terrestris, encoding an enzyme that metabolises the N-cyanoamidine neonicotinoids thiacloprid and acetamiprid with high efficiency. The genomic location and complete ORF of CYP9Q6 was corroborated by PCR and its metabolic activity characterised in vitro by expression in an insect cell line. CYP9Q6 metabolises both thiacloprid and acetamiprid more rapidly than the previously reported CYP9Q4 and CYP9Q5. We further demonstrate a direct, in vivo correlation between the expression of the CYP9Q6 enzyme in transgenic Drosophila melanogaster and an increased tolerance to thiacloprid and acetamiprid. We conclude that CYP9Q6 is an efficient metaboliser of N-cyanoamidine neonicotinoids and likely plays a key role in the high tolerance of B. terrestris to these insecticides.