Insecticide Susceptibilities and Enzyme Activities of Four Stink Bug Populations in Mississippi, USA.

In Mississippi, the Pentatomidae complex infesting soybean is primarily composed of Euschistus servus, Nezara viridula, Chinavia hilaris, and Piezodorus guildinii. This study employed spray bioassays to evaluate the susceptibilities of these stink bugs to seven commonly used formulated insecticides: oxamyl, acephate, bifenthrin, λ-cyhalothrin, imidacloprid, thiamethoxam, and sulfoxaflor. Stinks bugs were collected from soybeans in Leland, MS, USA during 2022 and 2023, as well as from wild host plants in Clarksdale, MS. There was no significant difference in the susceptibility of C. hilaris to seven insecticides between two years, whereas P. guildinii showed slightly increased susceptibility to neonicotinoids in 2023. Among all four stink bug species, susceptibility in 2022 was ranked as P. guildinii ≤ C. hilaris ≈ N. viridula, while in 2023, it was ranked as P. guildinii ≤ C. hilaris ≤ E. Servus. Additionally, populations of E. servus and P. guildinii collected from Clarksdale exhibited high tolerance to pyrethroids and neonicotinoids. Moreover, populations of E. servus and P. guildinii from SIMRU-2022 and Clarksdale-2023 showed elevated esterase and cytochrome P450 activity, respectively. These findings from spray bioassays and enzyme activity analyses provide a baseline for monitoring insecticide resistance in Pentatomidae and can guide insecticide resistance management strategies for Mississippi soybean.

Microarray and Functional Pathway Analyses Revealed Significantly Elevated Gene Expressions Associated with Metabolic Resistance to Oxamyl (Vydate) in Lygus lineolaris.

The tarnished plant bug (TPB, Lygus lineolaris) remains a major pest for a variety of crops. Frequent sprays on row crops, especially cotton, prompted resistance development in field populations. To maintain chemical control as an effective tool against the pest, knowledge of global gene regulations is desirable for better understanding and managing the resistance. Novel microarray expressions of 6688 genes showed 685 significantly upregulated and 1382 significantly downregulated genes in oxamyl-selected TPBs (Vyd1515FF[R]) from a cotton field. Among the 685 upregulated genes (participated in 470 pathways), 176 genes code 30 different enzymes, and 7 of the 30 participate in 24 metabolic pathways. Six important detoxification pathways were controlled by 20 genes, coding 11 esterases, two P450s, two oxidases, and three pathway-associated enzymes (synthases, reductase, and dehydrogenase). Functional analyses showed substantially enhanced biological processes and molecular functions, with hydrolase activity as the most upregulated molecular function (controlled by 166 genes). Eleven esterases belong to the acting on ester bond subclass of the 166 hydrolases. Surprisingly, only one GST showed significant upregulation, but it was not involved in any detoxification pathway. Therefore, this research reports a set of 20 genes coding 6 enzyme classes to detoxify a carbamate insecticide oxamyl in Vyd1515FF. Together with three previous reports, we have obtained the best knowledge of resistance mechanisms to all four conventional insecticide classes in the economically important crop pest. This valuable finding will greatly facilitate the development of molecular tools to monitor and manage the resistance and to minimize risk to environment.

Resistance risk assessment of six pyrethroids and acephate toward the resistant adult tarnished plant bug, Lygus lineolaris.

Due to rapidly developed resistance, pest management relies less on pyrethroids to control economically damaging infestations of the tarnished plant bug (TPB), Lygus lineolaris (Palisot de Beauvois) in cotton fields of Mississippi. Yet, pyrethroid resistance remains prevalent in TPB populations. This study assessed the resistance levels in adult TPB to six common pyrethroids and acephate. Resistant TBPs were collected from wild host plants in late October after harvest in the Mississippi Delta region of the United States. Based on LC50 values, the field-resistant TPBs displayed higher resistance to permethrin, esfenvalerate, and bifenthrin (approximately 30 fold) and moderate resistance to λ-cyhalothrin, ß-cyfluthrin, ζ-cypermethrin, and acephate (approximately 15 fold). Further investigations showed that the inhibitors of three detoxification enzyme, triphenyl phosphate (TPP), diethyl maleate (DEM), and piperonyl butoxide (PBO) had synergistic effects on permethrin, λ-cyhalothrin, and bifenthrin in resistant TPBs. Furthermore, elevated esterase, GST, and P450 activities were significantly expressed in field-resistant TPBs. Additionally, GST and esterase were reduced after 48 h exposure to certain pyrethroids at LC50 dose. The synergistic and biochemical assays consistently indicated that P450 and esterase were involved in pyrethroid detoxification in TPBs. This study provides valuable information for the continued use of pyrethroids and acephate in controlling TPBs in cotton fields in the Mississippi Delta region of the United States.

Toxicity Assessment of Four Formulated Pyrethroid-Containing Binary Insecticides in Two Resistant Adult Tarnished Plant Bug (Lygus lineolaris) Populations.

Over the past several decades, the extensive use of pyrethroids has led to the development of resistance in many insect populations, including the economically damaging pest tarnished plant bug (TPB), Lygus lineolaris, on cotton. To manage TPB resistance, several commercially formulated pyrethroid-containing binary mixtures, in combination with neonicotinoids or avermectin are recommended for TPB control and resistance management in the mid-South USA. This study aimed to evaluate the toxicity and resistance risks of four formulated pyrethroid-containing binary mixtures (Endigo, Leverage, Athena, and Hero) on one susceptible and two resistant TPB populations, which were field-collected in July (Field-R1) and October (Field-R2), respectively. Based on LC50 values, both resistant TPB populations displayed variable tolerance to the four binary mixtures, with Hero showing the highest resistance and Athena the lowest. Notably, the Field-R2 exhibited 1.5-3-fold higher resistance compared to the Field-R1 for all four binary insecticides. Moreover, both resistant TPB populations demonstrated significantly higher resistance ratios towards Hero and Leverage compared to their corresponding individual pyrethroid, while Endigo and Athena showed similar or lower resistance. This study also utilized the calculated additive index (AI) and co-toxicity coefficient (CTC) analysis, which revealed that the two individual components in Leverage exhibited antagonist effects against the two resistant TPB populations. In contrast, the two individual components in Endigo, Hero, and Athena displayed synergistic interactions. Considering that Hero is a mixture of two pyrethroids that can enhance the development of TPB resistance, our findings suggest that Endigo and Athena are likely superior products for slowing down resistance development in TPB populations. This study provides valuable insight for selecting the most effective mixtures to achieve better TPB control through synergistic toxicity analysis, while simultaneously reducing economic and environmental risks associated with resistance development in the insect pest.

The mechanisms of metabolic resistance to pyrethroids and neonicotinoids fade away without selection pressure in the tarnished plant bug Lygus lineolaris.

BACKGROUND: Heavy selection pressure prompted the development of resistance in a serious cotton pest tarnished plant bug (TPB), Lygus Lineolaris in the mid-southern United States. Conversely, a laboratory resistant TPB strain lost its resistance to five pyrethroids and two neonicotinoids after 36 generations without exposure to any insecticide. It is worthwhile to examine why the resistance diminished in this population and determine whether the resistance fade away has practical value for insecticide resistance management in TPB populations. RESULTS: A field-collected resistant TPB population in July (Field-R1) exhibited 3.90-14.37-fold resistance to five pyrethroids and two neonicotinoids, while another field-collected TPB population in April (Field-R2) showed much lower levels of resistance (0.84-3.78-fold) due to the absence of selection pressure. Interestingly, after 36 generations without exposure to insecticide, the resistance levels in the same population [laboratory resistant strain (Lab-R)] significantly decreased to 0.80-2.09-fold. The use of detoxification enzyme inhibitors had synergistic effects on permethrin, bifenthrin and imidacloprid in resistant populations of Lygus lineolaris. The synergism was more pronounced in Field-R2 than laboratory susceptible (Lab-S) and Lab-R TPB population. Moreover, esterase, glutathione S-transferase (GST), and cytochrome P450-monooxygenases (P450) enzyme activities increased significantly by approximately 1.92-, 1.43-, and 1.44-fold in Field-R1, respectively, and 1.38-fold increased P450 enzyme activities in Field-R2 TPB population, compared to the Lab-S TPB. In contrast, the three enzyme activities in the Lab-R strain were not significantly elevated anymore relative to the Lab-S population. Additionally, Field-R1 TPB showed elevated expression levels of certain esterase, GST and P450 genes, respectively, while Field-R2 TPB overexpressed only P450 genes. The elevation of these gene expression levels in Lab-R expectedly diminished to levels close to those of the Lab-S TPB populations. CONCLUSION: Our results indicated that the major mechanism of resistance in TPB populations was metabolic detoxification, and the resistance development was likely conferred by increased gene expressions of esterase, GST, and P450 genes, the fadeaway of the resistance may be caused by reversing the overexpression of esterase, GST and P450. Without pesticide selection, resistant gene (esterase, GST, P450s) frequencies declined, and detoxification enzyme activities returned to Lab-S level, which resulted in the recovery of the susceptibility in the resistant TPB populations. Therefore, pest's self-purging of insecticide resistance becomes strategically desirable for managing resistance in pest populations. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.

Detect Cytochrome C Oxidase- and Glutathione-S-Transferase-Mediated Detoxification in a Permethrin-Resistant Population of Lygus lineolaris.

Frequent sprays on cotton prompted resistance development in the tarnished plant bug (TPB). Knowledge of global gene regulation is highly desirable to better understand resistance mechanisms and develop molecular tools for monitoring and managing resistance. Novel microarray expressions of 6688 genes showed 3080 significantly up- or down-regulated genes in permethrin-treated TPBs. Among the 1543 up-regulated genes, 255 code for 39 different enzymes, and 15 of these participate in important pathways and metabolic detoxification. Oxidase is the most abundant and over-expressed enzyme. Others included dehydrogenases, synthases, reductases, and transferases. Pathway analysis revealed several oxidative phosphorylations associated with 37 oxidases and 23 reductases. One glutathione-S-transferase (GST LL_2285) participated in three pathways, including drug and xenobiotics metabolisms and pesticide detoxification. Therefore, a novel resistance mechanism of over-expressions of oxidases, along with a GST gene, was revealed in permethrin-treated TPB. Reductases, dehydrogenases, and others may also indirectly contribute to permethrin detoxification, while two common detoxification enzymes, P450 and esterase, played less role in the degradation of permethrin since none was associated with the detoxification pathway. Another potential novel finding from this study and our previous studies confirmed multiple/cross resistances in the same TPB population with a particular set of genes for different insecticide classes.

Role of DSC1 in Drosophila melanogaster synaptic activities in response to haedoxan A.

Drosophila sodium channel 1 (DSC1) encodes a voltage-gated divalent cation channel that mediates neuronal excitability in insects. Previous research revealed that DSC1 knockout Drosophila melanogaster conferred different susceptibility to insecticides, which indicated the vital regulation role of DSC1 under insecticide stress. Haedoxan A (HA) is a lignan compound isolated from Phryma leptostachya, and we found that HA has excellent insecticidal activity and is worthy of further study as a botanical insecticide. Herein, we performed bioassay and electrophysiological experiments to test the biological and neural changes in the larval Drosophila with/without DSC1 knockout in response to HA. Bioassay results showed that knockout of DSC1 reduced the sensitivity to HA in both w1118 (a common wild-type strain in the laboratory) and parats1 (a pyrethroid-resistant strain) larvae. Except for parats1 /DSC1-/- , electrophysiology results implicated that HA delayed the decay rate and increased the frequency of miniature excitatory junctional potentials of Drosophila from w1118 , parats1 , and DSC1-/- strains. Moreover, the neuromuscular synapse excitatory activities of parats1 /DSC1-/- larvae were more sensitive to HA than DSC1-/- larvae, which further confirmed the functional contribution of DSC1 to neuronal excitability. Collectively, these results indicated that the DSC1 channel not only regulated the insecticidal activity of HA, but also maintained the stability of neural circuits through functional interaction with voltage-gated sodium channels. Therefore, our study provides useful information for elucidating the regulatory mechanism of DSC1 in the neural system of insects involving the action of HA derived from P. leptostachya.

Fire ants feed their nestmates with their own venom.

Venom secretion is widely used by ants for disease control and more generally as an external surface disinfectant. Here we report evidence that Solenopsis invicta feed their nestmates with their own venom. Venom alkaloids were found in crops and midguts of ants at concentration levels that have previously been reported as effective against various pathogens. These venom alkaloids were found in midguts of the larvae, indicating that trophallaxis must be involved in the transfer of venom, since larvae do not produce alkaloids and they depend on workers to be fed. After the mating flight, the female alates shed their wings, burrow into the soil, and start new colonies. The new queen provided alkaloids to her first batch of larvae in the new colony. Since the crops of female alates contain venom alkaloids donated from their nestmate workers, the transfer of worker alkaloids to new generation occurred. After minim adult workers emerged, they took the role in providing venom to the larvae in the colony. Minim adult workers eventually died out and the normal workers became the venom donors in the colony. Although other functions may be possible, considering the well-known antimicrobial property of venom alkaloids and their detected concentration levels, venom in the digestive system is most likely used as an internal antibiotic by fire ants.

Characterization of two kdr mutations at predicted pyrethroid receptor site 2 in the sodium channels of Aedes aegypti and Nilaparvata lugens.

Pyrethroid insecticides prolong the opening of insect sodium channels by binding to two predicted pyrethroid receptor sites (PyR), PyR1 and PyR2. Many naturally-occurring sodium channel mutations that confer pyrethroid resistance (known as knockdown resistance, kdr) are located at PyR1. Recent studies identified two new mutations, V253F and T267A, at PyR2, which co-exist with two well-known mutations F1534C or M918T, at PyR1, in pyrethroid-resistant populations of Aedes aegypti and Nilaparvata lugens, respectively. However, the role of the V253F and T267A mutations in pyrethroid resistance has not been functionally examined. Here we report functional characterization of the V253F and T267A mutations in the Ae. aegypti sodium channel AaNav2-1 and the N. lugens sodium channel NlNav1 expressed in Xenopus oocytes. Both mutations alone reduced channel sensitivity to pyrethroids, including etofenprox. We docked etofenprox in a homology model of the pore module of the NlNav1 channel based on the crystal structure of an open prokaryotic sodium channel NavMs. In the low-energy binding pose etofenprox formed contacts with V253, T267 and a previously identified L1014 within PyR2. Combining of V253F or T267A with F1534C or M918T results in a higher level of pyrethroid insensitivity. Furthermore, both V253F and T267A mutations altered channel gating properties. However, V253F- and T267A-induced gating modifications was not observed in the double mutant channels. Our findings highlight the first example in which naturally-found combinational mutations in PyR1 and PyR2 not only confer higher level pyrethroid insensitivity, but also reduce potential fitness tradeoff in pyrethroid-resistant mosquitoes caused by kdr mutation-induced sodium channel gating modifications.

Incorporation of Poly(Ionic Liquid) with PVDF-HFP-Based Polymer Electrolyte for All-Solid-State Lithium-Ion Batteries.

A solid-state polymer electrolyte membrane is formed by blending poly(vinylidene fluoride-co-hexafluoropropylene) with the synthesized copolymer of poly(methyl methacrylate-co-1-vinyl-3-butyl-imidazolium bis(trifluoromethanesulfonyl)imide, in which lithium bis(trifluoromethane)sulfonimide molecules are applied as the source of lithium ions. The accordingly formed membrane that contains 14 wt.% of P(MMA-co-VBIm-TFSI), 56 wt.% of PVDF-HFP, and 30 wt.% of LiTFSI manifests the best electrochemical properties, achieving an ionic conductivity of 1.11 × 10-4 S·cm-1 at 30 °C and 4.26 × 10-4 S·cm-1 at 80 °C, a Li-ion transference number of 0.36, and a wide electrochemical stability window of 4.7 V (vs. Li/Li+). The thus-assembled all-solid-state lithium-ion battery of LiFePO4/SPE/Li delivers a discharge specific capacity of 148 mAh·g-1 in the initial charge-discharge cycle at 0.1 C under 60 °C. The capacity retention of the cell is 95.2% after 50 cycles at 0.1 C and the Coulombic efficiency remains close to 100% during the cycling process.

Surfactant affects the tool use behavior of foraging ants.

Surfactants are commonly used in detergents, soaps and agrichemical products. After use, the residual surfactants can be dispersed into environmental compartments, directly or indirectly affecting aquatic and terrestrial organisms. Ants are one of the few insects that are able to make and use tools when foraging for liquid food. However, this unique behavior of ants may be greatly affected by environmental pollutants. Here, we hypothesized that surfactants have adversarial impacts on ant foraging behavior, and tested this hypothesis by investigating the effect of TWEEN 80 (a common nonionic surfactant) on the tool use behavior of black imported fire ants (Solenopsis richteri) when foraging for liquid food (sugar water). Natural pine needles and man-made sponges were provided as tools for ants. The results revealed increasing surfactant concentration induced ants to deposit more tools and caused a higher drowning rate of ants. S. richteri tended to deposit more pine needles and tools of smaller size when exposed to surfactant. Interactions between tool type and surfactant concentration showed significant effects on tool deposition and drowning rate of ants. Addition of surfactant into sugar water increased the drowning rate and reduced the foraging activity and food collection of ant workers, suggesting that surfactant in liquid food can affect the foraging efficiency of ants. However, availability of tools reduced drowning rate and increased sugar water collected compared to without tools. Our results demonstrated that ants can adjust their tool use strategies to manage the foraging risk caused by environmental surfactant, such as increasing the amount and selecting appropriate size of the tools and assembling tools of different structures. Therefore, long-term exposure to surfactants may alter foraging behavior of ants and contribute to evolve new foraging strategy.

Negative cross-resistance of a pyrethroid-resistant Drosophila mutant to Phryma leptostachya-derived haedoxan A.

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Mutations in sodium channel confer knockdown resistance (kdr) to pyrethroids in various arthropod pests. Haedoxan A (HA) is the major insecticidal component from Phryma leptostachya. It has been shown that HA alters electrical responses at the Drosophila neuromuscular junction and modifies the gating properties of cockroach sodium channels expressed in Xenopus oocytes. However, whether sodium channel mutations that confer pyrethroid resistance also affect the action of HA is unknown. In this study, we conducted bioassays using HA and permethrin in two Drosophila melanogaster strains: w1118 , an insecticide-susceptible strain, and parats1 , a pyrethroid-resistant strain due to a I265N mutation in the sodium channel, and identified a new case of negative cross-resistance (NCR) between permethrin and HA. Both parats1 larvae and adults were more resistant to permethrin, as expected. However, both parats1 larvae and adults were more sensitive to HA compared to w1118 . We confirmed that the I265N mutation reduced the sensitivity to permethrin of a Drosophila sodium channel variant, DmNav 22, expressed in Xenopus oocytes. Interestingly, the I265N mutation also abolished the effect of HA on sodium channels. Further characterization showed that I265 on the sodium channels is critical for the action of both pyrethroids and HA on sodium channels, pointing to an overlapping mode of action between pyrethroids and HA on the sodium channel. Overall, our results suggest an I265N-independnt mechanism(s) in parats1 flies that is responsible for the NCR between permethrin and HA at the whole insect level.

The Odorant Binding Protein, SiOBP5, Mediates Alarm Pheromone Olfactory Recognition in the Red Imported Fire Ant, Solenopsis invicta.

Olfaction is crucial in mediating various behaviors of social insects such as red imported fire ants, Solenopsis invicta Buren. Olfactory receptor (OR) complexes consist of odor-specific ORs and OR co-receptors (Orco). Orcos are highly conserved across insect taxa and are widely co-expressed with ORs. Odorant binding proteins (OBPs) can transport semiochemicals to ORs as protein carriers and thus constitute the first molecular recognition step in insect olfaction. In this study, three OBP genes highly expressed in S. invicta antenna, OBP1, OBP5, OBP6, and Orco were partially silenced using RNA interference (RNAi). RNAi SiOBP5- and Orco-injected ants showed significantly lower EAG (electroantennography) responses to fire ant alarm pheromones and the alkaloid, 2,4,6-trimethylpyridine than water- or GFP-injected ants 72 h post injection. Subsequent qRT-PCR analysis demonstrated that the transcript level of the OBP1, OBP5, OBP6, and Orco significantly decreased 72 h after ants were injected with dsRNAs; however, there were no transcript level or EAG changes in ants fed dsRNAs. Our results suggest that S. invicta Orco and SiOBP5 are crucial to fire ants for their responses to alarm pheromones. RNAi knocking down SiOBP5 can significantly disrupt alarm pheromone communication, suggesting that disrupting SiOBP5 and Orcos could be potentially useful in the management of red imported fire ants.

Charge substitutions at the voltage-sensing module of domain III enhance actions of site-3 and site-4 toxins on an insect sodium channel.

Scorpion α-toxins bind at the pharmacologically-defined site-3 on the sodium channel and inhibit channel inactivation by preventing the outward movement of the voltage sensor in domain IV (IVS4), whereas scorpion ß-toxins bind at site-4 on the sodium channel and enhance channel activation by trapping the voltage sensor of domain II (IIS4) in its outward position. However, limited information is available on the role of the voltage-sensing modules (VSM, comprising S1-S4) of domains I and III in toxin actions. We have previously shown that charge reversing substitutions of the innermost positively-charged residues in IIIS4 (R4E, R5E) increase the activity of an insect-selective site-4 scorpion toxin, Lqh-dprIT3-c, on BgNav1-1a, a cockroach sodium channel. Here we show that substitutions R4E and R5E in IIIS4 also increase the activity of two site-3 toxins, LqhαIT from Leiurusquinquestriatus hebraeus and insect-selective Av3 from Anemonia viridis. Furthermore, charge reversal of either of two conserved negatively-charged residues, D1K and E2K, in IIIS2 also increase the action of the site-3 and site-4 toxins. Homology modeling suggests that S2-D1 and S2-E2 interact with S4-R4 and S4-R5 in the VSM of domain III (III-VSM), respectively, in the activated state of the channel. However, charge swapping between S2-D1 and S4-R4 had no compensatory effects on gating or toxin actions, suggesting that charged residue interactions are complex. Collectively, our results highlight the involvement of III-VSM in the actions of both site 3 and site 4 toxins, suggesting that charge reversing substitutions in III-VSM allosterically facilitate IIS4 or IVS4 voltage sensor trapping by these toxins.

Mapping the interaction surface of scorpion ß-toxins with an insect sodium channel.

The interaction of insect-selective scorpion depressant ß-toxins (LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus) with the Blattella germanica sodium channel, BgNav1-1a, was investigated using site-directed mutagenesis, electrophysiological analyses, and structural modeling. Focusing on the pharmacologically defined binding site-4 of scorpion ß-toxins at the voltage-sensing domain II (VSD-II), we found that charge neutralization of D802 in VSD-II greatly enhanced the channel sensitivity to Lqh-dprIT3. This was consistent with the high sensitivity of the splice variant BgNav2-1, bearing G802, to Lqh-dprIT3, and low sensitivity of BgNav2-1 mutant, G802D, to the toxin. Further mutational and electrophysiological analyses revealed that the sensitivity of the WT = D802E < D802G < D802A < D802K channel mutants to Lqh-dprIT3 correlated with the depolarizing shifts of activation in toxin-free channels. However, the sensitivity of single mutants involving IIS4 basic residues (K4E = WT << R1E < R2E < R3E) or double mutants (D802K = K4E/D802K = R3E/D802K > R2E/D802K > R1E/D802K > WT) did not correlate with the activation shifts. Using the cryo-EM structure of the Periplaneta americana channel, NavPaS, as a template and the crystal structure of LqhIT2, we constructed structural models of LqhIT2 and Lqh-dprIT3-c in complex with BgNav1-1a. These models along with the mutational analysis suggest that depressant toxins approach the salt-bridge between R1 and D802 at VSD-II to form contacts with linkers IIS1-S2, IIS3-S4, IIIP5-P1 and IIIP2-S6. Elimination of this salt-bridge enables deeper penetration of the toxin into a VSD-II gorge to form new contacts with the channel, leading to increased channel sensitivity to Lqh-dprIT3.

Identification of multiple odorant receptors essential for pyrethrum repellency in Drosophila melanogaster.

Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. In this study, we found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated our identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-ß-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. Our study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.

A dual-target molecular mechanism of pyrethrum repellency against mosquitoes.

Pyrethrum extracts from flower heads of Chrysanthemum spp. have been used worldwide in insecticides and repellents. While the molecular mechanisms of its insecticidal action are known, the molecular basis of pyrethrum repellency remains a mystery. In this study, we find that the principal components of pyrethrum, pyrethrins, and a minor component, (E)-ß-farnesene (EBF), each activate a specific type of olfactory receptor neurons in Aedes aegypti mosquitoes. We identify Ae. aegypti odorant receptor 31 (AaOr31) as a cognate Or for EBF and find that Or31-mediated repellency is significantly synergized by pyrethrin-induced activation of voltage-gated sodium channels. Thus, pyrethrum exerts spatial repellency through a novel, dual-target mechanism. Elucidation of this two-target mechanism may have potential implications in the design and development of a new generation of synthetic repellents against major mosquito vectors of infectious diseases.

Olfactory and behavioral responses to acetate esters in red imported fire ant, Solenopsis invicta.

BACKGROUND: The red imported fire ant, Solenopsis invicta Buren, is one of the most successful invasive ants in the world. Previous studies indicated that benzyl acetate and prenyl acetate elicited significant electroantennography (EAG) response in S. invicta and exhibited as attractants at certain concentrations. In addition, the easy commercial availability, low cost, and low mammalian toxicity make acetate esters ideal candidates to screen for potential use in S. invicta control. RESULTS: We examined the EAG and behavioral responses to 26 acetate esters in all castes of S. invicta. Our results demonstrated a large diversity of EAG tuning and behavioral responses to acetate esters in S. invicta. For linear alkyl acetate esters, EAG response was clearly affected by the carbon chain length of the alkyl group. Linear alkyl acetates with five to seven carbon chain length of the alkyl group elicited significant EAG response in S. invicta, whereas those with shorter (C1 to C4) or longer (C8 to C12) carbon chain lengths did not. Different substitutions also exhibited large variety of EAG and behavioral responses in S. invicta. CONCLUSION: Our works explored the olfactory and behavioral response of S. invicta to structurally different acetate esters, and identified two potential fire ant attractants, pentyl acetate and tran-2-hexenyl acetate, and two potential repellents, hexyl acetate and cis-3-hexenyl acetate. These compounds may be useful in developing new products for fire ant management. Published 2020. This article is a U.S. Government work and is in the public domain in the USA.

Chronology of sodium channel mutations associated with pyrethroid resistance in Aedes aegypti.

Aedes aegypti is the primary mosquito vector of dengue, yellow fever, Zika and chikungunya. Current strategies to control Ae. aegypti rely heavily on insecticide interventions. Pyrethroids are a major class of insecticides used for mosquito control because of their fast acting, highly insecticidal activities and low mammalian toxicity. However, Ae. aegypti populations around the world have begun to develop resistance to pyrethroids. So far, more than a dozen mutations in the sodium channel gene have been reported to be associated with pyrethroid resistance in Ae. aegypti. Co-occurrence of resistance-associated mutations is common in pyrethroid-resistant Ae. aegypti populations. As global use of pyrethroids in mosquito control continues, new pyrethroid-resistant mutations keep emerging. In this microreview, we compile pyrethroid resistance-associated mutations in Ae. aegypti in a chronological order, as they were reported, and summarize findings from functional evaluation of these mutations in an in vitro sodium channel expression system. We hope that the information will be useful for tracing possible evolution of pyrethroid resistance in this important human disease vector, in addition to the development of methods for global monitoring and management of pyrethroid resistance in Ae. aegypti.

Insecticidal and Enzyme Inhibitory Activities of Isothiocyanates against Red Imported Fire Ants, Solenopsis invicta.

Contact and fumigation toxicity of four isothiocyanates (ITCs), including allyl isothiocyanate (AITC), 3-butenyl isothiocyanate (3BITC), 3-(methylthio) propyl isothiocyanate (3MPITC) and 2-phenylethyl isothiocyanate (2PEITC), were evaluated against the red imported fire ant worker, Solenopsis invicta Buren. 2PEITC and 3MPITC exhibited strong contact toxicity. The median lethal dose (LD50)value of AITC, 2PEITC and 3MPITC were 7.99, 2.36 and 2.09 µg/ant respectively. In addition, AITC and 3MPITC also showed strong fumigation toxicity but not 2PEITC. The median lethal concentration (LC50) values of AITC and 3MPITC were 32.49 and 57.6 µg/L, respectively. In contrast, 3BITC did not exhibit any contact and fumigation toxicity even at 100 µg/µL. Esterase (EST), glutathione S-transferase (GST) and acetylcholinesterase (AChE)-inhibiting activities were assessed for three ITCs in S. invicta workers. All three ITCs inhibited both EST and GST activities but not AChE. The in vitro half maximal inhibitory concentration (IC50)values of AITC, 2PEITC and 3MPITC for GST were 3.32, 0.61 and 0.66 µg/µL, respectively. These results suggested that naturally occurring ITCs might be potentially useful for developing fire ants control products.