Varroa destructor is a major threat to honey bee health, and resistance to the limited number of approved synthetic miticides continues to increase worldwide. Identifying safe synergists that enhance the efficacy of existing acaricides is therefore a critical priority. We evaluated the toxicity, repellency, and synergistic potential of trans-chrysanthemic acid (TCA), a natural product terpenoid component of pyrethrins, against V. destructor and honey bees. To V. destructor, TCA had low contact toxicity (24 h LC50 = 155 mg/L) but coapplication of TCA with τ-fluvalinate significantly (P < 0.05) increased toxicity by 5.4-fold when compared to τ-fluvalinate alone. TCA also exhibited significant (P < 0.05) repellency in laboratory assays, though repellency did not translate to field conditions. To honey bees, TCA was found to have low toxicity via oral and topical exposure and co-treatment with τ-fluvalinate did not induce mortality or upregulate detoxification or stress marker genes. In a simulated hive assay using mites attached to bees, the TCA/fluvalinate combination resulted in 85% corrected mortality at 48 h, exceeding the sum of individual treatments and confirming synergism. Electrophysiological recordings of the V. destructor synganglion demonstrated TCA significantly increased fluvalinate-induced neuronal firing when compared to τ-fluvalinate alone, providing a mechanistic basis for the observed synergy. Together, these findings indicate that TCA is a safe and effective synergist that enhances τ-fluvalinate toxicity to V. destructor minimal acute toxicity to honey bees under the conditions tested, supporting its potential as a supplemental tool for V. destructor management.

Vector-borne diseases represent a significant global health concern, and effective vector control in animals often involves using orally administered drugs that kill arthropod vectors of human pathogens. Isoxazoline ectoparasiticides may have promise in humans if they can be optimized for safe use due to their selectivity for invertebrate over mammalian ion channels. Yet, isoxazolines can cause neurological side effects due to their ability to cross the blood brain barrier, and thus, we synthesized novel isoxazolines with improved physiochemical properties to reduce brain exposure without reducing toxicity to arthropod pests. Our medicinal chemistry campaigns led to the discovery of lead compound mCMV280 that is 3× more toxic to ticks and equitoxic to mosquitoes, with an ∼5× reduction in mammalian brain exposure and an ∼8× lower brain-to-plasma ratio compared to fluralaner. These findings highlight the promise of new isoxazoline scaffolds for safer and more effective drug-based vector control strategies in humans.

Varroa destructor is an obligate ectoparasite and a major contributor to global colony losses of western honey bees (Apis mellifera L.). Salivary secretions that modulate host immune responses and facilitate feeding are critical for successful parasitism of bee hosts, yet gaps in knowledge regarding the functional relevance of individual secreted proteins to feeding and parasitism remain. In this study, integrated proteomic and transcriptomic analyses were conducted to characterize the sialome of V. destructor and to identify salivary proteins involved in feeding physiology. Among the highly expressed candidates, five proteins, VdOBP2, VdAV422, VTP, VdCHIsal, and VdKazal, were selected for further investigation. Tissue-specific expression of these genes was quantified by qPCR, revealing abundant transcription in the salivary glands of both mobile and reproductive mites. RNAi-mediated knockdown of these genes did not affect mite survival but knockdown of VdAV422, VTP, and VdCHIsal significantly reduced mite ingestion. Furthermore, knockdown of VdAV422 and VdCHIsal markedly decreased mite fertility. These data demonstrate that specific salivary proteins are essential for efficient feeding and reproduction of V. destructor. The comprehensive proteome-transcriptome dataset of the salivary gland and secreted saliva combined with the functional assays provide a valuable foundation for defining 1) V. destructor-bee interactions, 2) mechanisms of V. destructor parasitism, and 3) technology development to reduce mite-feeding induced damage.

Huanglongbing (HLB), caused by the phloem-restricted bacterium Candidatus Liberibacter asiaticus (C. Las), is vectored by the Asian citrus psyllid (ACP) and threatens global citrus production. We demonstrate that VU041 (C19H20F3N3O), a small-molecule inhibitor of inward rectifier potassium (Kir) channels, provides a dual-mechanism approach to management of Huanglongbing (HLB) disease by combining acute toxicity with antifeedant activity. Topical and tarsal contact bioassays revealed that VU041 induced rapid mortality to ACP (24 h LD50 = 25 ng/insect) and reduced salivary gland secretion to suppress feeding behaviors. In semifield acquisition assays on C. Las-infected citrus, foliar application of VU041 significantly reduced both ACP survival (up to 96% at 365 ppb) and C. Las acquisition in surviving ACP. Combined, these results indicate that VU041 represents a promising chemical lead for developing insecticides that simultaneously kill ACP vectors and block pathogen acquisition at sublethal concentrations, representing a potentially transformative approach to reducing HLB-mediated losses in citrus production.

Fipronil is a widely used phenylpyrazole insecticide with an environmental and mammalian safety profile that limits its use in agricultural systems, including livestock production. Ethiprole, an alternative phenylpyrazole insecticide, is used in crop protection because of its more favorable safety profile but it is not currently utilized in livestock systems. The aim of this study was to evaluate the toxicity of ethiprole in insecticide susceptible and multi-insecticide resistant strains of the house fly (Musca domestica), a major pest in numerous livestock systems. A secondary aim was to assess the effects of an Rdl target-site mutation on ethiprole using Drosophila melanogaster. Ethiprole and fipronil were essentially equivalent in toxicity in both topical and feeding assays for both strains of M. domestica. Compared to fipronil, ethiprole elicited a significantly greater increase in firing frequency during the rising phase of nerve excitation in larval CNS of susceptible M. domestica, but only at the highest concentration tested (10 μM). In a glass contact assay, the Rdl mutant D. melanogaster had a 50.3-fold resistance ratio compared to the susceptible strain with ethiprole, while only 3.7-fold was observed with fipronil. Recovery of nerve activity from GABA-induced silencing in larval D. melanogaster CNS was more pronounced in the Rdl mutant than the susceptible strain and dependent on insecticide and concentration. These findings are in support of ethiprole being pursued as an M. domestica control insecticide in animal agriculture with some caution should an Rdl mutation arise in M. domestica populations.

The global rise of mosquito-borne diseases and widespread resistance to existing insecticides highlight the urgent need for novel, field-relevant mosquitocides. Here, we report the development and validation of a high-throughput, in vivo screening assay capable of evaluating adult mosquito toxicity across large chemical libraries. Utilizing a 96-well plate format, this assay enables simultaneous testing of hundreds of compounds per run using both net and filter paper substrates, with direct measurement of adult mosquito knockdown and mortality via tarsal contact - an exposure route highly relevant to real-world vector control tools such as long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS). The workflow is compatible with both manual and automated (robotic) liquid handling, with robotic dispensing yielding LC50 values within 2- to 3-fold of manual methods, thereby supporting scalability and sensitivity. Screening throughput is significantly enhanced, condensing weeks of testing into a half-day and enabling the evaluation of thousands of compounds per week. The assay rapidly identifies "hit" compounds for further formulation and field evaluation and is adaptable to multiple mosquito species. This platform represents a transformative advance for mosquitocide discovery, directly addressing key bottlenecks in vector control and resistance management.

We tested the toxicological profile and influence to feeding behaviors of novel N-arylamide compounds to the green peach aphid, Myzus persicae. Compounds 1 (C9H3Cl2F7N2O) and 2 (C10H3Cl2F8NO) are highly toxic with 4-h LC50 values of 44 ng/mL and 54 ng/mL, respectively via leaf dip bioassays. In addition to toxicity, altering probing behavior of aphids is an advantageous end point as specific behaviors are correlated to acquisition and transmission of plant viruses. Thus, we aimed to test if 1 and 2 altered probing and feeding behavior via electrical penetration graph (EPG) recordings and 1 and 2 significantly (P < 0.01) increased the time to first probe, the time required for initialization of piercing and sampling plant cell contents, and reduced initiation of new probes. Next, we aimed to facilitate systemic movement of 1 and 2 through plant tissues using a natural product based solubilizer containing rubusoside. Indeed, solubilization of 1 and 2 enabled systemic movement of compounds throughout the plant as evidenced by high aphid toxicity. Together, 1 and 2 represent novel aphicide scaffolds that can provide a dual approach to controlling economic losses stemming from aphid feeding by inducing mortality at concentrations similar to commercialized aphicides and alter probing behaviors relevant to virus transmission.

This study explored how dilution protocols, enzymes, and solvents affected the performance of 2- and 3-phenyl substituted methylcarbamates with varying selectivity for Anopheles gambiae acetylcholinesterase (AgAChE). Protocol A was 100-fold suspension in buffer of a 0.1 M DMSO stock solution, followed by serial buffer dilutions to give declining DMSO in parallel with the inhibitor. Protocol B was identical to A except that the initial stock concentration was 0.01 M. Protocol C entailed DMSO serial dilutions of a 0.1 M DMSO stock solution, followed by suspension of each into buffer giving 0.1 % (v/v) DMSO in all incubates. An. gambiae enzymes from insect homogenates or a recombinant clone generally showed a progressive increase in Hill slope from 0.5 to 1 via protocols A-C, along with increased IC50 values, with the 3-tert-butyl analog (1) epitomizing these effects. In contrast, propoxur displayed no consistent change in inhibition potency of any AChE, regardless of DMSO dilution procedure. DMSO at constant 35 μM or 0.000273 % had a midpoint effect on compound 1 inhibition and displayed competitive inhibition. Time course incubations (10-60 min) over a broad concentration range (10-12 - 10-5 M) of 1 revealed saturable inhibition of high and low potency, with the high potency effect more sensitive to incubation time. In contrast, there was little change of inhibition potency or Hill slope for compound 1 with human AChE, or the AChEs of Drosophila melanogaster and Musca domestica under any DMSO dilution protocol. Moreover, when the dilution protocols were repeated using ethanol as a solvent, little change of inhibition potency or Hill slope was observed with any compound, enzyme, or dilution scheme. These results contradicted the expectation that higher solvent concentration would yield better solubility and more rapid and potent effects of these lipophilic insecticides. Molecular modeling suggests DMSO may be competing with carbamate binding to AgAChE or by stabilizing an allosteric subpocket within AgAChE.

There is a paucity of information pertaining to the fundamental roles of glia in insect central nervous system (CNS) function and in the maintenance of ionic gradients. Inward rectifier potassium (Kir) channels are known to drive K+ buffering in mammals, but cellular expression patterns and the physiological roles of neural or glial Kir channels in insects have remained undefined. We show that Kir2 channels are expressed in astrocyte-like glia (ALG) and subperineurial glia, and live-cell imaging indicated that ALG Kir channels are involved in Drosophila CNS function by providing a mechanism for K+ buffering. The inhibition of Drosophila ALG Kir channels reduced K+ buffering events and altered spontaneous neural firing changes to Drosophila behavior. These data confirm insect glia are active participants supporting CNS function and highlight a conserved evolutionary mechanism for K+ homeostasis in neural systems across mammals and insects.

Leptospermone, a natural β-triketone and major constituent of manuka oil (Leptospermum scoparium), is an established inhibitor of plant HPPD and was identified to induce rapid knockdown and induce high toxicity to Aedes aegypti adults via topical and tarsal contact exposure with LD50 values of 150 ng/mg of mosquito and 357 ng/cm2, respectively. Although toxic to mosquitoes, leptospermone was non-toxic to ticks, the honey bee, or the fruit fly indicating a high degree of insect specificity. Importantly, leptospermone was equally toxic to non-blood fed and blood-fed mosquitoes suggesting the mode of action is not via HPPD inhibition. Molecular modeling suggested high structural similarities between leptospermone and mammalian sulfonamide carbonic anhydrase (CA) inhibitors. In vitro potency assays with mosquito midgut homogenate or purified CA verify leptospermone inhibits Ae. aegypti CA, but not mammalian CAs. CAs are metalloenzymes that regulate the pH of tissues and ubiquitously expressed throughout insect tissues but are abundantly expressed in the mosquito midgut and, thus, we tested leptospermone to alter pH regulation in the mosquito midgut. Indeed, leptospermone significantly reduced the pH of Ae. aegypti midguts when compared to control mosquitoes which further supports the notion that leptospermone mode of action in insects is via inhibition of CA. These data verify leptospermone is an effective mosquitocide that induces rapid knockdown and toxicity to Ae. aegypti at doses that approach natural pyrethrins against pyrethroid-resistant mosquito strains. Further, the data indicate leptospermone mode of action is CA inhibition, which is a novel mosquitocide target and is different when compared to the mode of action in plants.

BACKGROUND:The devastating honey bee (Apis mellifera) pest, Varroa destructor, has developed resistance to commonly used synthetic acaricides, such as amitraz, tau-fluvalinate, and coumaphos. To find new active ingredients that may be useful in reducing V. destructor populations in honey bee colonies, we examined the acute toxicity of isoxazoline insecticides that are toxic to ectoparasites, such as ticks and fleas. RESULTS:Here, we evaluated the toxicity of afoxolaner, fluralaner, sarolaner, and lotilaner to V. destructor and honey bees using direct application methods. Fluralaner (median lethal dose (LD50) = 0.07 ng/V. destructor) was the most toxic isoxazoline insecticide and only 2× less toxic than amitraz (0.04 ng/V. destructor), but 25,600× more toxic than coumaphos (1789 ng/V. destructor). Sarolaner (selectivity ratio = 0.3) was much more toxic to honey bees than to V. destructor, while afoxolaner toxicity was equal to mites and bees. Fluralaner and lotilaner were 123× and 2× more toxic to V. destructor than to honey bees. In addition to describing isoxazolines as a putative chemical class to control V. destructor, we tested the potency of fluralaner against the firing frequency of V. destructor central neurons. Exposure to 30 μm fluralaner led to reversal of GABA inhibition with a significant increase of nerve firing when compared to GABA firing rates. CONCLUSION:These data suggest that fluralaner represents a potential candidate for V. destructor control in colonies due to its high toxicity to V. destructor and its relatively low toxicity to honey bees. Additionally, methodological details for electrophysiological recordings on V. destructor central nervous system (CNS) firing rates can be used to advance the development of new miticides. © 2025 Society of Chemical Industry.

Short-term exposure of mosquitoes to insecticides offers a valuable opportunity to identify early molecular responses that underpin toxicity and resistance. Using 3' RNA-sequencing, we investigated genotype-specific transcriptomic changes in pyrethroid-susceptible (Rockefeller; ROCK) and -resistant (Puerto Rico; PR) Aedes aegypti following short-term exposure (30 min) to the pyrethroid insecticide deltamethrin. The ROCK strain exhibited minimal transcriptional remodeling, and few differentially expressed genes (DEGs), suggesting limited acute sensitivity at the tested dose. In contrast, the PR strain displayed 67 DEGs enriched in pathways related to insecticide metabolism, energy homeostasis, and neuroactive ligand-receptor interactions. Consistent with known resistance mechanisms, the PR strain showed strong constitutive and inducible expression of detoxification-related cytochrome P450 (CYP) genes, including CYP6BB2, CYP6M11, and CYP4C38, as well as upregulation of CYP9J family genes linked to resistance loci in comparison to ROCK. Additional molecular distinctions highlighted altered cuticle synthesis, diverse neuroreceptor expression patterns, including GPCRs and glutamate receptors and epigenetic signatures, such as histone downregulation and reduced DNMT2 expression, suggesting enhanced transcriptional flexibility in the PR strain. Gene co-expression analyses supported greater metabolic adaptability in PR mosquitoes compared to ROCK. Together, these findings reveal that resistance to deltamethrin in Ae. aegypti is not only mediated by detoxification enzymes and reduced cuticular permeability but also involves neuromodulatory and epigenetic components. This work underscores the utility of short-term transcriptomic profiling for uncovering insecticide response mechanisms and highlights candidate genes for functional validation. These insights can guide the design of next-generation insecticides and resistance management interventions.

Abstract Background The Lone Star tick, Amblyomma americanum is important to human health because of a variety of pathogenic organisms transmitted to humans during feeding events, which underscores the need to identify novel approaches to prevent tick bites. Thus, the goal of this study was to test natural and synthetic molecules for repellent activity against ticks in spatial, contact and human fingertip bioassays. Methods The efficacy of essential oils and naturally derived compounds as repellents to Am. americanum nymphs was compared in three different bioassays: contact, spatial and fingertip repellent bioassays. Results Concentration response curves after contact exposure to 1R-trans-chrysanthemic acid (TCA) indicated a 5.6 μg/cm2 concentration required to repel 50% of ticks (RC50), which was five- and sevenfold more active than DEET and nootkatone, respectively. For contact repellency, the rank order of repellency at 50 μg/cm2 for natural oils was clove > geranium > oregano > cedarwood > thyme > amyris > patchouli > citronella > juniper berry > peppermint > cassia. For spatial bioassays, TCA was approximately twofold more active than DEET and nootkatone at 50 μg/cm2 but was not significantly different at 10 μg/cm2. In spatial assays, thyme and cassia were the most active compounds tested with 100% and 80% ticks repelled within 15 min of exposure respectively and was approximately twofold more effective than DEET at the same concentration. To translate these non-host assays to efficacy when used on the human host, we quantified repellency using a finger-climbing assay. TCA, nootkatone and DEET were equally effective in the fingertip assay, and patchouli oil was the only natural oil that significantly repelled ticks. Conclusions The differences in repellent potency based on the assay type suggests that the ability to discover active tick repellents suitable for development may be more complicated than with other arthropod species; furthermore, the field delivery mechanism must be considered early in development to ensure translation to field efficacy. TCA, which is naturally derived, is a promising candidate for a tick repellent that has comparable repellency to commercialized tick repellents. Graphical Abstract

The dinoflagellate Karenia brevis is a causative agent of red tides in the Gulf of Mexico and generates a potent family of structurally related brevetoxins that act via the voltage-sensitive Na+ channel. This project was undertaken to better understand the neurotoxicology and kdr cross-resistance to brevetoxins in house flies by comparing the susceptible aabys strain to ALkdr (kdr) and JPskdr (super-kdr). When injected directly into the hemocoel, larvae exhibited rigid, non-convulsive paralysis consistent with prolongation of sodium channel currents, the known mechanism of action of brevetoxins. In neurophysiological studies, the firing frequency of susceptible larval house fly central nervous system preparations showed a > 200% increase 10 min after treatment with 1 nM brevetoxin-3. This neuroexcitation is consistent with the spastic paralytic response seen after hemocoel injections. Target site mutations in the voltage-sensitive sodium channel of house flies, known to confer knockdown resistance (kdr and super-kdr) against pyrethroids, attenuated the effect of brevetoxin-3 in baseline firing frequency and toxicity assays. The rank order of sensitivity to brevetoxin-3 in both assays was aabys > ALkdr > JPskdr. At the LD50 level, resistance ratios for the knockdown resistance strains were 6.9 for the double mutant (super-kdr) and 2.3 for the single mutant (kdr). The data suggest that knockdown resistance mutations may be one mechanism by which flies survive brevetoxin-3 exposure during red tide events.

Tick serine protease inhibitors (serpins) play crucial roles in tick feeding and pathogen transmission. We demonstrate that Ixodes scapularis (Ixs) nymph tick saliva serpin (S) 41 (IxsS41), secreted by Borrelia burgdorferi (Bb)-infected ticks at high abundance, is involved in regulating tick evasion of host innate immunity and promoting host colonization by Bb. Recombinant (r) proteins were expressed in Pichia pastoris, and substrate hydrolysis assays were used to determine. Ex vivo (complement and hemostasis function related) and in vivo (paw edema and effect on Bb colonization of C3H/HeN mice organs) assays were conducted to validate function. We demonstrate that rIxsS41 inhibits chymase and cathepsin G, pro-inflammatory proteases that are released by mast cells and neutrophils, the first immune cells at the tick feeding site. Importantly, stoichiometry of inhibition analysis revealed that 2.2 and 2.8 molecules of rIxsS41 are needed to 100% inhibit 1 molecule of chymase and cathepsin G, respectively, suggesting that findings here are likely events at the tick feeding site. Furthermore, chymase-mediated paw edema, induced by the mast cell degranulator, compound 48/80 (C48/80), was blocked by rIxsS41. Likewise, rIxsS41 reduced membrane attack complex (MAC) deposition via the alternative and lectin complement activation pathways and dose-dependently protected Bb from complement killing. Additionally, co-inoculating C3H/HeN mice with Bb together with rIxsS41 or with a mixture (rIxsS41 and C48/80). Findings in this study suggest that IxsS41 markedly contributes to tick feeding and host colonization by Bb. Therefore, we conclude that IxsS41 is a potential candidate for an anti-tick vaccine to prevent transmission of the Lyme disease agent.

Background The cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a destructive agricultural pest, capable of photosynthate removal and plant virus transmission. Therefore, we aimed to test the antifeedant properties of small-molecule inhibitors of inward rectifier potassium (Kir) channels expressed in insect salivary glands and develop an approach for enabling systemic movement of lipophilic Kir inhibitors. Results Two Kir channel inhibitors, VU041 and VU730, reduced the secretory activity of the aphid salivary glands by 3.3-fold and foliar applications of VU041 and VU730 significantly (P < 0.05) increased the time to first probe, total probe duration, and nearly eliminated phloem salivation and ingestion. Next, we aimed to facilitate systemic movement of VU041 and VU730 through evaluation of a novel natural product based solubilizer containing rubusoside that was isolated from Chinese sweet leaf (Rubus suavissimus) plants. A single lower leaf was treated with Kir inhibitor soluble liquid (KI-SL) and systemic movement throughout the plant was verified via toxicity bioassays and changes to feeding behavior through the electrical penetration graph (EPG) technique. EPG data indicate KI-SL significantly reduced ability to reach E1 (phloem salivation) and E2 (phloem ingestion) waveforms and altered plant probing behavior when compared to the untreated control. High-performance liquid chromatography (HPLC) analysis indicated the presence of VU041 and VU730 in the upper leaf tissue of these plants. Together, these data provide strong support that incorporation of rubusoside with Kir inhibitors enhanced translaminar and translocation movement through the plant tissue. Conclusion These data further support hemipteran Kir channels as a target to prevent feeding and induce toxicity. Further, these studies highlight a novel delivery approach for generating plant systemic activity of lipophilic insecticides. (c) 2022 Society of Chemical Industry.

Declines in managed honey bee populations are multifactorial but closely associated with reduced virus immunocompetence and thus, mechanisms to enhance immune function are likely to reduce viral infection rates and increase colony viability. However, gaps in knowledge regarding physiological mechanisms or ‘druggable’ target sites to enhance bee immunocompetence has prevented therapeutics development to reduce virus infection. Our data bridge this knowledge gap by identifying ATP-sensitive inward rectifier potassium (K ATP ) channels as a pharmacologically tractable target for reducing virus-mediated mortality and viral replication in bees, as well as increasing an aspect of colony-level immunity. Bees infected with Israeli acute paralysis virus and provided K ATP channel activators had similar mortality rates as uninfected bees. Furthermore, we show that generation of reactive oxygen species (ROS) and regulation of ROS concentrations through pharmacological activation of K ATP channels can stimulate antiviral responses, highlighting a functional framework for physiological regulation of the bee immune system. Next, we tested the influence of pharmacological activation of K ATP channels on infection of 6 viruses at the colony level in the field. Data strongly support that K ATP channels are a field-relevant target site as colonies treated with pinacidil, a K ATP channel activator, had reduced titers of seven bee-relevant viruses by up to 75-fold and reduced them to levels comparable to non-inoculated colonies. Together, these data indicate a functional linkage between K ATP channels, ROS, and antiviral defense mechanisms in bees and define a toxicologically relevant pathway that can be used for novel therapeutics development to enhance bee health and colony sustainability in the field.

Inhibitors targeting the 4-hydroxyphenyl pyruvate dioxygenase (HPPD) enzyme are well established herbicides and HPPD is also a primary enzyme within the tyrosine metabolism pathway in hematophagous arthropods, which is an essential metaboilic pathway post-blood feeding to prevent tyrosine-mediated toxicity. The objective of this study was to characterize the toxicity of triketone, pyrazole, pyrazolone, isoxazole, and triazole herbicides that inhibit HPPD to blood-fed mosquitoes and ticks. Topical exposure of nitisinone to blood-fed Aedes aegypti yielded high toxicity with an LD50 of 3.81 ng/insect (95% CI: 3.09 to 4.67 ng; Hillslope: 0.97, r2: 0.99), yet was non-toxic to non-blood fed (NBF) mosquitoes. The rank order of toxicity was nitisinone > tembotrione > pyrazoxyfen > tebuconazole > mesotrione against blood-fed Ae. Aegypti, but nitisinone was approximately 30-fold more toxic than other chemicals tested. We also assessed the toxicity of HPPD-inhibiting herbicides to the lone star tick, Amblyomma americanum and similarly, nitisinone was toxic to Am. americanum with a lethal time to kill 50% of subjects (LT50) of 23 h at 10 μM. Knockdown of the gene encoding the HPPD enzyme was performed through RNA-interference led to significant mortality after blood feeding in both, Ae. aegypti and Am. americanum. Lastly, a fluorescence assay was developed to determine relative quantities of L-tyrosine in Ae. aegypti and Am. americanum treated with HPPD inhibitors. L-tyrosine levels correlated with toxicity with nitisinone exposure leading to increased tyrosine concentrations post-blood feeding. Taken together, these data support previous work suggesting HPPD-inhibitors represent a novel mode of toxicity to mosquitoes and ticks and may represent base scaffolds for development of novel insecticides specific for hematophagous arthropods.

Mosquito-borne diseases are a significant threat to human health. The frequent and repetitive application of insecticides can result in the selection of resistant mosquito populations leading to product failures for reducing community disease transmission. It is important that new interventions are discovered and developed for reducing mosquito populations and, in turn, protecting human health. Plant essential oils are promising chemical interventions for reducing mosquito populations. The myrtle family, Myrtaceae, has numerous species to be studied as potential bioinsecticides. Here, we combined toxicological, biochemical, and neurophysiological ap-proaches to provide evidence for cajeput oil and terpene constituents to elicit bioinsecticidal activity to pyrethroid-susceptible and-resistant Aedes aegypti. We show cajeput oil terpenes to enhance cAMP production, increase ACh levels, inhibit in vivo and in vitro AChE activity, and disrupt spike discharge frequencies of the mosquito CNS. This study presents the first report on the bioinsecticidal activity of cajeput oil terpenes to pyrethroid-susceptible and-resistant mosquitoes and provides comparative data for the octopaminergic system as a putative molecular target for the bioinsecticides with implications for resistance management.

The K+/Cl- cotransporter (KCC) is the primary mechanism by which mature neurons maintain low intracellular chloride (Cl-) concentration and has been shown to be functionally coupled to the GABA-gated chloride channels (GGCC) in Drosophila central neurons. Further, pharmacological inhibition of KCC has been shown to lead to acute toxicity of mosquitoes that highlights the toxicological relevance of insect KCC. Yet, gaps in knowledge remain regarding physiological drivers of KCC function and interactions of ion flux mechanisms upstream of GGCC in insects. Considering this, we employed electrophysiological and fluorescent microscopy techniques to further characterize KCC in the insect nervous system. Fluorescent microscopy indicated insect KCC2 is expressed in rdl neurons, which is the neuron type responsible for GABA-mediated neurotransmission, and are coexpressed with inward rectifier potassium (Kir) 2 channels. Coexpression of Kir2 and KCC2 suggested the possibility of functional coupling between these two K+ flux pathways. Indeed, extracellular recordings of Drosophila CNS showed pre-block of Kir channels prior to block of KCC led to a significant (P < 0.001) increase in CNS firing rates over baseline that when taken together, supports functional coupling of Kir to KCC function. Additionally, we documented a synergistic increase to toxicity of VU0463271, an established KCC inhibitor, above the expected additive toxicity after co-treatment with the Kir inhibitor, VU041. These data expand current knowledge regarding the physiological roles of KCC and Kir channels in the insect nervous system by defining additional pathways that facilitate inhibitory neurotransmission through GGCC.