Previous studies have shown that blockers of voltage-gated potassium (Kv) channels, such as 4-aminopyridine (4-AP) and 2-methoxy-N-((1-phenylcyclopentyl)methyl)benzamide (2-MPB) synergized pyrethroid ...toxicity as well, or better than, piperonyl butoxide. The present study assessed the involvement of different Kv channels as possible pyrethroid synergist targets in Drosophila melanogaster. Three Kv1 mutants (Sh5, Sh133, and ShM) and one Kv2 mutant (Shab3) were tested. All Kv1 mutant flies showed increased sensitivity to permethrin in topical and glass contact toxicity assays, of 2- to 11-fold. Central nervous system (CNS) recordings of larval D. melanogaster showed a similar pattern of increased sensitivity. Potentiated effects were also observed with deltamethrin on the mutants Sh5 (30- to 35-fold) and Sh133 (33- to 47-fold), but the mutant ShM showed little change in sensitivity. In contrast, the Shab3 strain showed toxicity and physiological effects of both pyrethroids that were similar to the susceptible OR strain. Thus, some K+ channel mutations mimicked the synergistic effect of channel blockers. Additional studies showed that Shab3 had the highest sensitivity to 4-AP in topical assays, and the Shaker-null mutants, ShM and Sh133 showed greater sensitivity to 2-MPB in CNS recordings of larval D. melanogaster. These results suggest that Kv1 channels are a useful synergist target for pyrethroids, as assessed both in whole insects and at the level of the nervous system. Thus, Kv1-targeting compounds can potentially serve as insect control tools to reduce pyrethroid use via synergistic action.
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•Kv1 mutant flies showed increased toxicity to permethrin.•Neuronal activity of mutants had a similar pattern of increased sensitivity.•The ShM and Shab3 mutants had less effect on permethrin activity.•Some K+ channel mutations mimicked the synergistic effect of channel blockers.•K+ channel blockers can be effective synergists of pyrethroids.
Ligand-gated chloride channels underlie inhibition in excitable membranes and are proven target sites for insecticides. The γ-aminobutyric acid (GABA1) receptor/chloride ionophore complex is the ...primary site of action for a number of currently used insecticides, such as lindane, endosulfan, and fipronil. These compounds act as antagonists by stabilizing nonconducting conformations of the chloride channel. Blockage of the GABA-gated chloride channel reduces neuronal inhibition, which leads to hyperexcitation of the central nervous system, convulsions, and death. We recently investigated the mode of action of the silphinenes, plant-derived natural compounds that structurally resemble picrotoxinin. These materials antagonize the action of GABA on insect neurons and block GABA-mediated chloride uptake into mouse brain synaptoneurosomes in a noncompetitive manner. In mammals, avermectins have a blocking action on the GABA-gated chloride channel consistent with a coarse tremor, whereas at longer times and higher concentrations, activation of the channel suppresses neuronal activity. Invertebrates display ataxia, paralysis, and death as the predominant signs of poisoning, with a glutamate-gated chloride channel playing a major role. Additional target sites for the avermectins or other chloride channel-directed compounds might include receptors gated by histamine, serotonin, or acetylcholine. The voltage-sensitive chloride channels form another large gene family of chloride channels. Voltage-dependent chloride channels are involved in a number of physiological processes including: maintenance of electrical excitability, chloride ion secretion and resorption, intravesicular acidification, and cell volume regulation. A subset of these channels is affected by convulsants and insecticides in mammals, although the role they play in acute lethality in insects is unclear. Given the wide range of functions that they mediate, these channels are also potential targets for insecticide development.
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Delivery of compounds to the brain is critical for the development of effective treatment therapies of multiple central nervous system diseases. Recently a novel insect-based brain ...uptake model was published utilizing a locust brain ex vivo system. The goal of our study was to develop a priori, in silico cheminformatic models to describe brain uptake in this insect model, as well as evaluate the predictive ability. The machine learning program Orange® was used to evaluate several machine learning (ML) models on a published data set of 25 known drugs, with in vitro data generated by a single laboratory group to reduce inherent inter-laboratory variability. The ML models included in this study were linear regression (LR), support vector machines (SVN), k-nearest neighbor (kNN) and neural nets (NN). The quantitative structure–property relationship models were able to correlate experimental logCtot (concentration of compound in brain) and predicted brain uptake of r2 > 0.5, with the descriptors log(P*MW−0.5) and hydrogen bond donor used in LR, SVN and KNN, while log(P*MW−0.5) and total polar surface area (TPSA) descriptors used in the NN models. Our results indicate that the locust insect model is amenable to data mining chemoinformatics and in silico model development in CNS drug discovery pipelines.
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•Novel method to record nerve firing of mosquito larvae central nervous system.•Larvae pinned and organs were removed to provide access to ventral nerve cord.•Electrode connected to ...axons of nerve cord allowed for high signal-to-noise ratio.•Allows for characterization of drugs/neurotransmitters on mosquito CNS.•Method can characterize insecticide resistance produced at the level of the nerve.
Resistance to currently utilized chemical insecticidal agents represents a significant threat to public health and food security worldwide. Better understanding the neurophysiological effects of available and candidate insecticidal molecules is valuable for characterizing the mechanisms of insecticide resistance, as well as the design and study of novel control chemistries. In this paper, we describe a method of recording nerve firing from the central nervous system of Aedes aegypti fourth instar larvae. In short, mosquito larvae were immobilized by placing small pins through the head and siphon of the larvae in a wax dish, ventral side down. A single, longitudinal, dorsal incision from the distal abdomen to the pronotum of the larva was made, the alimentary canal removed, and the ventral nerve cord severed between the second and third abdominal ganglia. A recording suction electrode was connected directly to axons within the severed end of the connective in a novel way to record nerve firing in the ventral nerve cord at a high signal-to-noise ratio with conventional electrophysiological equipment. Using this novel method, we report the effects of four neuroactive compounds using this method: octopamine, pilocarpine, nicotine, and γ-aminobutyric acid (GABA). The utility of this recording technique for elucidating target site mechanisms involved in insecticide resistance is demonstrated with p,p’-dichlorodiphenyltrichlorethane (DDT) and its difluoro analog (DFDT).
The emergence of pyrethroid-resistant mosquitoes is a worldwide problem that necessitates further research into the development of new repellents and insecticides. This study explored the ...modification of existing pyrethroid acids to identify structural motifs that might not be affected by kdr active site mutations that elicit pyrethroid resistance. Because synthetic pyrethroids almost always contain activity-dependent chiral centers, we chose to focus our efforts on exploring alkoxy moieties of esters obtained with 1R-trans-permethrinic and related acids, which we showed in previous studies to have repellent and/or repellent synergistic properties. To this end, compounds were synthesized and screened for spatially acting repellency and insecticidal activity against the susceptible, Orlando, and pyrethroid-resistant, Puerto Rico, strains of Aedes aegypti mosquito. Screening utilized a high-throughput benchtop glass tube assay, and the compounds screened included a mixture of branched, unbranched, aliphatic, halogenated, cyclic, non-cyclic, and heteroatom-containing esters. Structure-activity relationships indicate that n-propyl, n-butyl, n-pentyl, cyclobutyl, and cyclopentyl substituents exhibited the most promising repellent activity with minimal kdr cross resistance. Preliminary testing showed that these small alcohol esters can be synergistic with phenyl amides and pyrethroid acids. Further derivatization of pyrethroid acids offer an interesting route to future active compounds, and while mosquitoes were the focus of this work, pyrethroid acids and esters have potential for use in reducing pest populations and damage in cropping systems as well.
Two new Amaryllidaceae alkaloids, belonging to the mesembrine- and crinine-types, named crinsarnine (
) and sarniensinol (
), were isolated from the dried bulbs of
together with bowdensine (
), ...sarniensine (
), hippadine (
) and 1-
-acetyl-lycorine (
). Crinsarnine (
) and sarniensinol (
) were characterized using spectroscopic and chiroptical methods as (1
,2
,4a
,10b
)-2,7-dimethoxy-1,2,3,4,4a,6-hexahydro-5,11b-ethano1,3dioxolo-4,5-
phenanthridin-1-yl acetate and (6-(3a
,4
,6
,7a
)-6-methoxy-1-methyl-2,3,3a,6,7,7a-hexa-hydro-1
-indol-3a-yl)benzo
1,3dioxol-5-yl)methanol, respectively. Furthermore, the complete spectroscopic characterization of bowdensine (
) is reported for the first time. Compounds
-
were evaluated against the Orlando reference strain of
. None of compounds showed mortality against 1st instar
larvae at the concentrations tested. In adult topical bioassays, only
displayed adulticidal activity with an LD
= 2.29 ± 0.049 μg/mosquito. As regards the structure-activity relationship, the pretazettine and crinine scaffold in
and
and in
and
respectively, proved to be important for their activity, while the pyrrole
phenanthridine scaffold present in
and
was important for their reactivity. Among the pretazettine group compounds, opening of the B ring or the presence of a B ring lactone as well as the
-stereochemistry of the A/B ring junction, appears to be important for activity, while in crinine-type alkaloids, the substituent at C-2 seems to play a role in their activity.
Mosquitoes, such as
and
, are important vectors of human diseases. Fluralaner, a recently introduced parasiticide, was evaluated as a mosquitocide in this study. On
and
fourth-instar larvae, ...fluralaner had 24-h LC
(lethal concentration for 50% mortality) values of 1.8 ppb and 0.4 ppb, respectively. Following topical application to adult
, fluralaner toxicity reached a plateau in about 3 days, with 1- and 3-day LD
(lethal dose for 50% mortality) values of 1.3 ng/mg and 0.26 ng/mg, suggesting a slowly developing toxicity. Fipronil outperformed fluralaner by up to 100-fold in adult topical, glass contact, and feeding assays on
. These data show that fluralaner does not have exceptional toxicity to mosquitoes in typical exposure paradigms. In electrophysiological recordings on
larval central nervous system, the effectiveness of fluralaner for restoring nerve firing after gamma-aminobutyric acid (GABA) treatment, a measure of GABA antagonism, was similar in susceptible Oregon-R and cyclodiene-resistant
-1675 strains, with EC
(half maximal effective concentration) values of 0.34 µM and 0.29 µM. Although this finding suggests low cross resistance in the presence of
, the moderate potency, low contact activity, and slow action of fluralaner argue against its use as an adult mosquitocide for vector control.
We explored the potential of two sodium channel activators, veratrine and aconitine, as both insecticides and synergists of natural pyrethrins (NP) on Aedes aegypti adults and larvae. Aconitine was ...more toxic than veratrine, with an LD50 of 157 ng/mg compared to 376 ng/mg, on the pyrethroid-susceptible Orlando strain, but only aconitine showed significant resistance in the pyrethroid-resistant Puerto Rico strain (RR = 14.6 in topical application and 8.8 in larval bioassay). When applied in mixtures with piperonyl butoxide (PBO) and NP, large synergism values were obtained on the Orlando strain. Aconitine + PBO mixture synergized NP 21.8-fold via topical adult application and 10.2-fold in larval bioassays, whereas veratrine + PBO synergized NP 5.3-fold via topical application and 30.5-fold in larval bioassays. Less synergism of NP was observed on the resistant Puerto Rico strain, with acontine + PBO synergizing NP only 4.1-fold in topical application (8-fold in larval bioassays) and veratrine + PBO synergizing NP 9.5-fold in topical application (13.3-fold in larval bioassays). When alkaloids were applied directly to the mosquito larval nervous system, veratrine was nearly equipotent on both strains, while aconitine was less active on pyrethroid-resistant nerve preparations (no block at 10 μM compared to block at 1 μM on the susceptible strain). The nerve blocking effect of NP was significantly synergized by both compounds on the pyrethroid-susceptible strain by about 10-fold, however only veratrine synergized NP block on the pyrethroid-resistant strain, also showing 10-fold synergism). These results highlight the potential of site II sodium channel activators as insecticides and their ability to synergize pyrethroids, which may extend the commercial lifetime of these chemistries so essential to public health vector control.
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•Veratrine and aconitine are capable of synergizing natural pyrethrins (NP) on adult and larvae of Aedes aegypti.•Both alkaloids synergized NP on pyrethroid-susceptible strain; only veratrine synergized NP on the resistant strain.•Veratrine was more potent than aconitine (by itself and as a synergist of NP) on cockroach nerve cords.•Co-application with PBO increased synergism multiplicatively and suggested that synergism occurred via novel mechanism.
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