Characterization of the honeybee AmNaV1 channel and tools to assess the toxicity of insecticides.
ABSTRACT: Pollination is important for both agriculture and biodiversity. For a significant number of plants, this process is highly, and sometimes exclusively, dependent on the pollination activity of honeybees. The large numbers of honeybee colony losses reported in recent years have been attributed to colony collapse disorder. Various hypotheses, including pesticide overuse, have been suggested to explain the disorder. Using the Xenopus oocytes expression system and two microelectrode voltage-clamp, we report the functional expression and the molecular, biophysical, and pharmacological characterization of the western honeybee's sodium channel (Apis Mellifera NaV1). The NaV1 channel is the primary target for pyrethroid insecticides in insect pests. We further report that the honeybee's channel is also sensitive to permethrin and fenvalerate, respectively type I and type II pyrethroid insecticides. Molecular docking of these insecticides revealed a binding site that is similar to sites previously identified in other insects. We describe in vitro and in silico tools that can be used to test chemical compounds. Our findings could be used to assess the risks that current and next generation pesticides pose to honeybee populations.
Project description:A homology model of the housefly voltage-gated sodium channel was developed to predict the location of binding sites for the insecticides fenvalerate, a synthetic pyrethroid, and DDT an early generation organochlorine. The model successfully addresses the state-dependent affinity of pyrethroid insecticides, their mechanism of action and the role of mutations in the channel that are known to confer insecticide resistance. The sodium channel was modelled in an open conformation with the insecticide-binding site located in a hydrophobic cavity delimited by the domain II S4-S5 linker and the IIS5 and IIIS6 helices. The binding cavity is predicted to be accessible to the lipid bilayer and therefore to lipid-soluble insecticides. The binding of insecticides and the consequent formation of binding contacts across different channel elements could stabilize the channel when in an open state, which is consistent with the prolonged sodium tail currents induced by pyrethroids and DDT. In the closed state, the predicted alternative positioning of the domain II S4-S5 linker would result in disruption of pyrethroid-binding contacts, consistent with the observation that pyrethroids have their highest affinity for the open channel. The model also predicts a key role for the IIS5 and IIIS6 helices in insecticide binding. Some of the residues on the helices that form the putative binding contacts are not conserved between arthropod and non-arthropod species, which is consistent with their contribution to insecticide species selectivity. Additional binding contacts on the II S4-S5 linker can explain the higher potency of pyrethroid insecticides compared with DDT.
Project description:Fenvalerate is a pyrethroid insecticide with rapid action, strong targeting, broad spectrum, and high efficiency. However, continued use of fenvalerate has resulted in its widespread presence as a pollutant in surface streams and soils, causing serious environmental pollution. Pesticide residues in the soil are closely related to food safety, yet little is known regarding the kinetics and metabolic behaviors of fenvalerate. In this study, a fenvalerate-degrading microbial strain, CD-9, isolated from factory sludge, was identified as Citrobacter freundii based on morphological, physio-biochemical, and 16S rRNA sequence analysis. Response surface methodology analysis showed that the optimum conditions for fenvalerate degradation by CD-9 were pH 6.3, substrate concentration 77 mg/L, and inoculum amount 6% (v/v). Under these conditions, approximately 88% of fenvalerate present was degraded within 72 h of culture. Based on high-performance liquid chromatography and gas chromatography-mass spectrometry analysis, ten metabolites were confirmed after the degradation of fenvalerate by strain CD-9. Among them, o-phthalaldehyde is a new metabolite for fenvalerate degradation. Based on the identified metabolites, a possible degradation pathway of fenvalerate by C. freundii CD-9 was proposed. Furthermore, the enzyme localization method was used to study CD-9 bacteria and determine that its degrading enzyme is an intracellular enzyme. The degradation rate of fenvalerate by a crude enzyme solution for over 30 min was 73.87%. These results showed that strain CD-9 may be a suitable organism to eliminate environmental pollution by pyrethroid insecticides and provide a future reference for the preparation of microbial degradation agents and environmental remediation.
Project description:The voltage-sensitive sodium (Na+) channel (Vssc) is the target site of pyrethroid insecticides. Pest insects develop resistance to this class of insecticide by acquisition of one or multiple amino acid substitution(s) in this channel. In Southeast Asia, two major Vssc types confer pyrethroid resistance in the dengue mosquito vector Aedes aegypti, namely, S989P+V1016G and F1534C. We expressed several types of Vssc in Xenopus oocytes and examined the effect of amino acid substitutions in Vssc on pyrethroid susceptibilities. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to permethrin by 100- and 25-fold, respectively, while S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to permethrin by 1100-fold. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to deltamethrin by 10- and 1-fold (no reduction), respectively, but S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to deltamethrin by 90-fold. These results imply that pyrethroid insecticides are highly likely to lose their effectiveness against A. aegypti if such a Vssc haplotype emerges as the result of a single crossing-over event; thus, this may cause failure to control this key mosquito vector. Here, we strongly emphasize the importance of monitoring the occurrence of triple mutations in Vssc in the field population of A. aegypti.
Project description:Worldwide, increasing numbers of insects have evolved resistance to a wide range of pesticides, which hampers their control in the field and, therefore, threatens agriculture. Members of the carboxylesterase and cytochrome P450 monooxygenase superfamilies are prominent candidates to confer metabolic resistance to pyrethroid insecticides. Both carboxylesterases and P450 enzymes have been shown to be involved in pyrethroid resistance in Australian Helicoverpa armigera, the noctuid species possessing by far the most reported resistance cases worldwide. However, specific enzymes responsible for pyrethroid resistance in field populations of this species have not yet been identified. Here, we show that the resistance toward fenvalerate in an Australian strain of H. armigera is due to a unique P450 enzyme, CYP337B3, which arose from unequal crossing-over between two parental P450 genes, resulting in a chimeric enzyme. CYP337B3 is capable of metabolizing fenvalerate into 4'-hydroxyfenvalerate, which exhibits no toxic effect on susceptible larvae; enzymes from the parental P450 genes showed no detectable fenvalerate metabolism. Furthermore, a polymorphic H. armigera strain could be bred into a susceptible line possessing the parental genes CYP337B1 and CYP337B2 and a resistant line possessing only CYP337B3. The exclusive presence of CYP337B3 in resistant insects of this strain confers a 42-fold resistance to fenvalerate. Thus, in addition to previously documented genetic mechanisms of resistance, recombination can also generate selectively advantageous variants, such as this chimeric P450 enzyme with an altered substrate specificity leading to a potent resistance mechanism.
Project description:Voltage-gated sodium channels are critical for electrical signaling in the nervous system. Pyrethroid insecticides exert their toxic action by modifying the gating of sodium channels. A valine to methionine mutation in the transmembrane segment 6 of domain I (IS6) of sodium channels from tobacco budworms (Heliothis virescens) has been shown to alter channel gating and reduce insect sodium channel sensitivity to pyrethroids. A valine to leucine substitution was subsequently reported in pyrethroid-resistant bedbug populations. Intriguingly, pyrethroid-resistant mammalian sodium channels possess an isoleucine at the corresponding position. To determine whether different substitutions at this position alter channel gating and confer pyrethroid resistance, we made valine to methionine, isoleucine or leucine substitutions at the corresponding position, V409, in a cockroach sodium channel and examined the gating properties and pyrethroid sensitivity of the three mutants in Xenopus oocytes. All three mutations reduced the channel sensitivity to three pyrethroids (permethrin, cismethrin and deltamethrin). V409M, but not V409I or V409L, caused 6-7mV depolarizing shifts in the voltage dependences of both activation and inactivation. V409M and V409L slowed channel activation kinetics and accelerated open-state deactivation kinetics, but V409I did not. Furthermore, the substitution of isoleucine with valine, but not with methionine nor leucine, at the corresponding position in a rat skeletal muscle sodium channel, rNav1.4, enhanced channel sensitivity to deltamethrin. Collectively, our study highlights an important role of residues at 409 in regulating not only sodium channel gating, but also the differential sensitivities of insect and mammalian sodium channels to pyrethroids.
Project description:Voltage-gated sodium channels are the primary target of pyrethroids, an important class of synthetic insecticides. Pyrethroids bind to a distinct receptor site on sodium channels and prolong the open state by inhibiting channel deactivation and inactivation. Recent studies have begun to reveal sodium channel residues important for pyrethroid binding. However, how pyrethroid binding leads to inhibition of sodium channel deactivation and inactivation remains elusive. In this study, we show that a negatively charged aspartic acid residue at position 802 (D802) located in the extracellular end of transmembrane segment 1 of domain II (IIS1) is critical for both the action of pyrethroids and the voltage dependence of channel activation. Charge-reversing or -neutralizing substitutions (K, G, or A) of D802 shifted the voltage dependence of activation in the depolarizing direction and reduced channel sensitivity to deltamethrin, a pyrethroid insecticide. The charge-reversing mutation D802K also accelerated open-state deactivation, which may have counteracted the inhibition of sodium channel deactivation by deltamethrin. In contrast, the D802G substitution slowed open-state deactivation, suggesting an additional mechanism for neutralizing the action of deltamethrin. Importantly, Schild analysis showed that D802 is not involved in pyrethroid binding. Thus, we have identified a sodium channel residue that is critical for regulating the action of pyrethroids on the sodium channel without affecting the receptor site of pyrethroids.
Project description:The extensive reliance on insecticides to control Aedes aegypti mosquitoes and disrupt transmission of dengue, chikungunya and Zika has fueled the emergence of widespread resistance to insecticides. Mismatch between the frequency of pyrethroid resistance in mosquitoes and the occurrence of pyrethroid-based insecticide applications for vector control is often hypothesized to be due to household use of commercial insecticide products. We experimentally quantified phenotypic and genotypic responses of four Ae. aegypti strains (three field, pyrethroid resistant, and one laboratory, pyrethroid susceptible) after exposure to two commonly used household aerosol insecticide products (a space spray and a residual spray formulation) containing pyrethroid active ingredients. Experiments were performed within homes of Mérida, Mexico. After exposure to the products, all three pyrethroid resistant field Ae. aegypti strains had significantly lower mortality rates (averaging 41% and 50% for the two products, respectively) than the controls (99%). Applying insecticides as surface sprays led to a significant increase in the frequency of I1016 kdr homozygotes in surviving Ae. aegypti, suggesting strong selection pressure for this allele. Given the large-scale use of household aerosol insecticide products in areas that are endemic for Ae. aegypti-transmitted diseases, their role as a pyrethroid resistance selection source, particularly when used as space sprays, should be taken into consideration when designing resistance management plans.
Project description:The toxicity of pesticides used in agriculture towards non-targeted organisms and especially pollinators has recently drawn the attention from a broad scientific community. Increased honeybee mortality observed worldwide certainly contributes to this interest. The potential role of several neurotoxic insecticides in triggering or potentiating honeybee mortality was considered, in particular phenylpyrazoles and neonicotinoids, given that they are widely used and highly toxic for insects. Along with their ability to kill insects at lethal doses, they can compromise survival at sublethal doses by producing subtle deleterious effects. In this study, we compared the bee's locomotor ability, which is crucial for many tasks within the hive (e.g. cleaning brood cells, feeding larvae…), before and after an acute sublethal exposure to one insecticide belonging to the two insecticide classes, fipronil and thiamethoxam. Additionally, we examined the locomotor ability after exposure to pyrethroids, an older chemical insecticide class still widely used and known to be highly toxic to bees as well. Our study focused on young bees (day 1 after emergence) since (i) few studies are available on locomotion at this stage and (ii) in recent years, pesticides have been reported to accumulate in different hive matrices, where young bees undergo their early development. At sublethal doses (SLD48h, i.e. causing no mortality at 48 h), three pyrethroids, namely cypermethrin (2.5 ng/bee), tetramethrin (70 ng/bee), tau-fluvalinate (33 ng/bee) and the neonicotinoid thiamethoxam (3.8 ng/bee) caused a locomotor deficit in honeybees. While the SLD48h of fipronil (a phenylpyrazole, 0.5 ng/bee) had no measurable effect on locomotion, we observed high mortality several days after exposure, an effect that was not observed with the other insecticides. Although locomotor deficits observed in the sublethal range of pyrethroids and thiamethoxam would suggest deleterious effects in the field, the case of fipronil demonstrates that toxicity evaluation requires information on multiple endpoints (e.g. long term survival) to fully address pesticides risks for honeybees. Pyrethroid-induced locomotor deficits are discussed in light of recent advances regarding their mode of action on honeybee ion channels and current structure-function studies.
Project description:Knockdown resistance (kdr) to pyrethroid insecticides has been attributed to point mutations in the para sodium channel gene in more than a half dozen insect pest species. In this study, we identified two novel para mutations in five highly resistant kdr-type German cockroach strains. The two mutations, from glutamic acid (E434) to lysine (K434) and from cysteine (C764) to arginine (R764), respectively, are located in the first intracellular linker connecting domains I and II. E434K is located near the beginning of the linker (closest to domain I), whereas C764R is found toward the end of the linker (closest to domain II). Two additional mutations from aspartic acid (D58) to glycine (G58), and from proline (P1880) to leucine (L1888), respectively, were found in one of the resistant strains. The four mutations coexist with the previously identified leucine to phenylalanine (L993F) kdr mutation in IIS6, and are present only in the highly resistant individuals of a given strain. These findings suggest that these mutations might be responsible for high levels of knockdown resistance toward pyrethroid insecticides in the German cockroach.
Project description:Synthetic pyrethroid-fenvalerate-is one of the most widespread toxic pollutants and has adverse effect on living systems. However, little is known about its biotransformation mechanism in different microorganisms. To elucidate the pathway that might be involved in the catabolism of fenvalerate, we used Bacillus flexus strain XJU-4 (3-nitrobenzoate degrading organism) as an ideal fenvalerate degrading bacterium. Thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry analysis results revealed that 3-phenoxybenzoate, protocatechuate, and catechol are the three main by-products of fenvalerate metabolism. Additionally, the bacterial cell-free enzymes showed the activities of fenvalerate hydrolyzing esterase, 3-phenoxybenzaldehyde dehydrogenase, 3-phenoxybenzoate dioxygenase, phenol hydroxylase, protocatechuate 2,3-dioxygenase and catechol-2,3-dioxygenase. Thus, in strain XJU-4, protocatechuate and catechol were further metabolized through meta-cleavage pathway. Moreover, laboratory-scale soil experiments results suggest that B. flexus strain XJU-4 is a suitable contender for bioremediation of pyrethroid fenvalerate-contaminated sites.