(-)-Adaline from the Adalia Genus of Ladybirds Is a Potent Antagonist of Insect and Specific Mammalian Nicotinic Acetylcholine Receptors.

Ladybird beetles (Coleoptera: Coccinellidae) possess strong chemical defences that are secreted in response to stress and are also found on the coating of eggs, which are rich in alkaloids that are responsible for their toxicity to other species. Recent studies have shown that alkaloids from several species of ladybird beetle can target nicotinic acetylcholine receptors (nAChRs) acting as receptor antagonists. Here, we have explored the actions of (-)-adaline, found in the 2-spot (Adalia bipunctata) and 10-spot (Adalia decempunctata) ladybirds, on both mammalian (α1ß1γδ, α7, α4ß2, α3ß4) and insect nAChRs using patch-clamp of TE671 cells and locust brain neurons natively expressing nAChRs, as well as two-electrode voltage clamp of Xenopus laevis oocytes recombinantly expressing nAChRs. All nAChR subtypes were antagonised by (-)-adaline in a time-dependent, voltage-dependent and non-competitive manner with the lowest IC50s at rat α3ß4 (0.10 µM) and locust neuron (1.28 µM) nAChRs, at a holding potential of -75 mV. The data imply that (-)-adaline acts as an open channel blocker of nAChRs.

Batrachotoxin, pyrethroids, and BTG 502 share overlapping binding sites on insect sodium channels.

Batrachotoxin (BTX), a steroidal alkaloid, and pyrethroid insecticides bind to distinct but allosterically coupled receptor sites on voltage-gated sodium channels and cause persistent channel activation. BTX presumably binds in the inner pore, whereas pyrethroids are predicted to bind at the lipid-exposed cavity formed by the short intracellular linker-helix IIS4-S5 and transmembrane helices IIS5 and IIIS6. The alkylamide insecticide (2E,4E)-N-(1,2-dimethylpropyl)-6-(5-bromo-2-naphthalenyl)-2,4-hexadienamide (BTG 502) reduces sodium currents and antagonizes the action of BTX on cockroach sodium channels, suggesting that it also binds inside the pore. However, a pyrethroid-sensing residue, Phe(3i17) in IIIS6, which does not face the pore, is essential for the activity of BTG 502 but not for BTX. In this study, we found that three additional deltamethrin-sensing residues in IIIS6, Ile(3i12), Gly(3i14), and Phe(3i16) (the latter two are also BTX-sensing), and three BTX-sensing residues, Ser(3i15) and Leu(3i19) in IIIS6 and Phe(4i15) in IVS6, are all critical for BTG 502 action on cockroach sodium channels. Using these data as constraints, we constructed a BTG 502 binding model in which BTG 502 wraps around IIIS6, probably making direct contacts with all of the above residues on the opposite faces of the IIIS6 helix, except for the putative gating hinge Gly(3i14). BTG 502 and its inactive analog DAP 1855 antagonize the action of deltamethrin. The antagonism was eliminated by mutations of Ser(3i15), Phe(3i17), Leu(3i19), and Phe(4i15) but not by mutations of Ile(3i12), Gly(3i14), and Phe(3i16). Our analysis revealed a unique mode of action of BTG 502, its receptor site overlapping with those of both BTX and deltamethrin.

An important role of a pyrethroid-sensing residue F1519 in the action of the N-alkylamide insecticide BTG 502 on the cockroach sodium channel.

Deltamethrin, a pyrethroid insecticide, and BTG 502, an alkylamide insecticide, target voltage-gated sodium channels. Deltamethrin binds to a unique receptor site and causes prolonged opening of sodium channels by inhibiting deactivation and inactivation. Previous (22)Na(+) influx and receptor binding assays using mouse brain synaptoneurosomes showed that BTG 502 antagonized the binding and action of batrachotoxin (BTX), a site 2 sodium channel neurotoxin. However, the effect of BTG 502 has not been examined directly on sodium channels expressed in Xenopus oocytes. In this study, we examined the effect of BTG 502 on wild-type and mutant cockroach sodium channels expressed in Xenopus oocytes. Toxin competition experiments confirmed that BTG 502 antagonizes the action of BTX and possibly shares a common receptor site with BTX. However, unlike BTX which causes persistent activation of sodium channels, BTG 502 reduces the amplitude of peak sodium current. A previous study showed that BTG 502 was more toxic to pyrethroid-resistant house flies possessing a super-kdr (knockdown resistance) mechanism than to pyrethroid-susceptible house flies. However, we found that the cockroach sodium channels carrying the equivalent super-kdr mutations (M918T and L1014F) were not more sensitive to BTG 502 than the wild-type channel. Instead, a kdr mutation, F1519I, which reduces pyrethroid binding, abolished the action of BTG 502. These results provide evidence the actions of alkylamide and pyrethroid insecticides require a common sodium channel residue.

Development of HPLC analytical protocols for quantification of artemisinin in biomass and extracts.

Quantification of artemisinin purity and amount in plant material and extracts to date has been characterized by a considerable inconsistency in values. This is likely to be due to the adoption of varied analytical procedures and use of inappropriate to the specific applications analytical techniques. In this paper we are attempting to further develop artemisinin analysis to the point where a universally acceptable reference method is available to the research and end-users communities. Thus, we have developed and validated an HPLC-RI method and optimized an HPLC-ELSD method. We used the gradient HPLC-UV method recommended by the current artemisinin monograph as a comparison for the method improvements presented herein, and show the limitations for its application scope. The data reported should help to allow more reliable laboratory analysis of artemisinin in both pure samples and in Artemisia annua extracts.

Molecular determinants on the insect sodium channel for the specific action of type II pyrethroid insecticides.

Pyrethroid insecticides are classified as type I or type II based on their distinct symptomology and effects on sodium channel gating. Structurally, type II pyrethroids possess an alpha-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids. Both type I and type II pyrethroids inhibit deactivation consequently prolonging the opening of sodium channels. However, type II pyrethroids inhibit the deactivation of sodium channels to a greater extent than type I pyrethroids inducing much slower decaying of tail currents upon repolarization. The molecular basis of a type II-specific action, however, is not known. Here we report the identification of a residue G(1111) and two positively charged lysines immediately downstream of G(1111) in the intracellular linker connecting domains II and III of the cockroach sodium channel that are specifically involved in the action of type II pyrethroids, but not in the action of type I pyrethroids. Deletion of G(1111), a consequence of alternative splicing, reduced the sodium channel sensitivity to type II pyrethroids, but had no effect on channel sensitivity to type I pyrethroids. Interestingly, charge neutralization or charge reversal of two positively charged lysines (Ks) downstream of G(1111) had a similar effect. These results provide the molecular insight into the type II-specific interaction of pyrethroids with the sodium channel at the molecular level.

Use of pyrethroid analogues to identify key structural features for enhanced esterase resistance in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae).

It has been reported previously that the major resistance mechanism to pyrethroid insecticides by the cotton bollworm Helicoverpa armigera (Hübner) in Australia is a consequence of overproduction of esterase isoenzymes. This paper reports structure-activity relationships that support such a view, based on in vivo bioassays conducted with a range of pyrethroid structures containing a variety of acid and alcohol moieties and the correlation with in vitro esterase inhibition assays against the same structures, and identifies the critical regions of the molecule with regard to esterase inhibition, and hence resistance. The implications of this work in terms of possible resistance management are evaluated and discussed.

Modelling insecticide-binding sites in the voltage-gated sodium channel.

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.

An alanine in segment 3 of domain III (IIIS3) of the cockroach sodium channel contributes to the low pyrethroid sensitivity of an alternative splice variant.

In a previous study, we showed that two alternative exons (G1 and G2 encoding IIIS3-S4) were involved in the differential sensitivity of two cockroach sodium channel splice variants, BgNa(v)1-1 and BgNa(v)2-1 (previously called KD1 and KD2), to deltamethrin, a pyrethroid insecticide (Tan, et al., 2002b. Alternative splicing of an insect sodium channel gene generates pharmacologically distinct sodium channels. J. Neurosci. 22, 5300-5309.). Here, we report the identification of an amino acid residue in exon G2 that contributes to the low deltamethrin sensitivity of BgNa(v)2-1. Replacement of A1356 in BgNa(v)2-1 with the corresponding V1356 in BgNa(v)1-1 enhanced the sensitivity of the BgNa(v)2-1 channel to deltamethrin by six-fold. Conversely, substitution of V1356 with A1356 in BgNa(v)1-1 produced a recombinant BgNa(v)1-1 channel that was 5-fold more resistant to deltamethrin. These results demonstrate that A1356 contributes to the low sensitivity of BgNa(v)2-1 to deltamethrin. A1356V substitution also shifted the voltage-dependence of activation by 10 mV in the hyperpolarizing direction. Possible mechanisms by which this amino acid change affects the action of pyrethroids on the sodium channel are discussed.

Response of economically important aphids to components of Hemizygia petiolata essential oil.

The essential oil of Hemizygia petiolata Ashby (Lamiaceae) contains high levels (>70%) of the sesquiterpene (E)-beta-farnesene, the alarm pheromone for many economically important aphid species. In order to test the suitability of H. petiolata oil as a source of (E)-beta-farnesene for use in new integrated aphid control strategies, behavioural responses of pest aphid species were studied in laboratory and field experiments. In alarm pheromone assays the peach-potato aphid, Myzus persicae Sulzer, and the pea aphid, Acyrthosiphon pisum (Harr), showed a lower level of response to the oil than expected given the high levels of (E)-beta-farnesene. It was shown that minor components in the oil, (+)-bicyclogermacrene and (-)-germacrene D, caused inhibition of the alarm response for M. persicae and A. pisum respectively. Nevertheless, in olfactometer studies the oil was directly repellent to A. pisum and the grain aphid, Sitobion avenae F. Sitobion avenae was not only repelled by (E)-beta-farnesene but also by (-)-germacrene D. Furthermore, although it was not directly repellent to M. persicae, the oil interfered with its attraction to host plant stimuli. In field plot experiments, numbers of A. pisum were significantly reduced in plots treated with a slow release formulation of the oil, when compared with control plots.

Effects of plant-derived compounds on larvae of a blow fly species that causes secondary myiases: laboratory studies.

No-choice and binary-choice bioassays were used to test the effect of a range of plant-derived insecticides on the behaviour of larvae of the black blow fly Phormia regina (Meigen). Azadirachtin (100 and 10 ppm), pyrethrum extract (10 ppm) and the naphthoquinone BTG 505 (1000 ppm) acted as deterrents for P. regina larvae. The mortality of larvae was reduced in instances where they moved away from a treated diet. Larvae given a choice were heavier, when compared with larvae reared exclusively on diets containing either azadirachtin (100 or 10 ppm) or the naphthoquinones, BTG 504 (1000 ppm) or BTG 505 (1000 ppm). The deterrent effect of azadirachtin, pyrethrum and the naphthoquinone BTG 505 together with their larvicidal activity, could be utilized in prophylaxis against myiasis infections due to larvae of Phormia regina.

A new insecticidal protolimonoid from Aegle marmelos.

Bioassay-directed fractionation of the ethyl acetate extracts of the stem bark of Aegle marmelos Correa (Rutaceae) afforded a new compound, named skimmiarepin C, along with skimmiarepin A. The latter is a known compound but isolation from A. marmelos is new. The new compound is a senecioate ester analogue of the latter. Full identification of the new compound was achieved using spectroscopic methods on the separated mono-acetate derivatives. Skimmiarepins A and C exhibit moderate insecticidal activity against Phaedon cochleariae and Musca domestica in comparison with natural pyrethrum extract. The two epimeric acetates of skimmiarepin C are both less active.

Mode of action and pesticidal activity of the natural product dunnione and of some analogues.

This paper reports the investigation of the insecticidal and fungicidal activity of dunnione, a natural product obtained inadvertently as a by-product of a synthesis programme. Dunnione exhibits no insecticidal activity but has an unusually broad spectrum of antifungal activity. In vitro and in vivo (preventative) activities were comparable to those of several long-established fungicides (eg carbendazim). However, in whole-plant assays, its eradicant activity was unexpectedly low, probably due to poor dose-transfer from leaf surface to fungus. The level of residual activity appears to be influenced by the formulation. Finally, its potential as a lead structure was assessed, and several analogues synthesised which exhibited high activity in the in vitro assays. Mode-of-action studies revealed that dunnione exerts its action primarily through initiation of redox cycling. This contrasts to the activity of BTG 505, the biochemical/chemical precursor, which does not initiate redox cycling but instead exhibits both insecticidal and fungicidal activity by inhibiting mitochondrial Complex III.

An investigation of the ionophoric characteristics of destruxin A.

Destruxin A, a cyclohexadepsipeptide related to the enniatins and beauvericin, exhibits ionophoric properties. Calcium ion mobilization across liposomal membrane barriers, for example, has been demonstrated using the calcium ion-sensitive dyes Arsenazo III and Fura-2. Initial molecular mechanics/molecular dynamics calculations indicate the potential for destruxin A to form a coordination complex with calcium in which the divalent cation is bound at the center of a sandwich formed by two molecules of destruxin A. This novel calcium ion binding may help explain the diverse biological effects exhibited by the destruxins.

Herbicidal action of 2-hydroxy-3-alkyl-1,4-naphthoquinones.

The main mode of herbicidal activity of 2-hydroxy-3-alkyl-1,4-naphthoquinones is shown to be inhibition of photosystem II (PSII). The herbicidal and in vitro activities have been measured and correlated with their (Log)octanol/water partition coefficients (Log Ko/w). The length of the 3-n-alkyl substituent for optimal activity differed between herbicidal and in vitro activity. The maximum in vitro activity was given by the nonyl to dodecyl homologues (Log Ko/w between 6.54 and 8.12), whereas herbicidal activity peaked with the n-hexyl compound (Log Ko/w = 4.95). The effect of chain branching was also investigated using isomeric pentyl analogues substituted at position 3. All exhibited similar levels of in vitro activities but herbicidal activities differed, albeit moderately, with the exception of one analogue that was much less phytotoxic. Other modes of action were also investigated using two representative compounds. They did not show any activity on photosystem I or mitochondrial complex I, or generate toxic oxygen radicals by redox cycling reactions. Only moderate activity was found against mitochondrial complex III from plants, in contrast to much higher corresponding activity using an insect enzyme.

Insecticidal 2-hydroxy-3-alkyl-1,4-naphthoquinones: correlation of inhibition of ubiquinol cytochrome c oxidoreductase (complex III) with insecticidal activity.

The insecticidal and in vitro activities of four homologous series of 2-hydroxy and acetoxy-3-substituted-1,4-naphthoquinones have been measured and correlated with their (Log) octanol/water partition coefficients (Log Ko/w). In vitro activity against mitochondrial complex III was only exhibited by 2-hydroxy-3-alkyl-1,4-naphthoquinones, indicating that the 2-acetoxy compounds act as proinsecticides. Good correlation was observed between in vivo activity against the two-spotted spider mite, Tetranychus urticae and inhibition of complex III isolated from blowfly flight muscle. Both hydroxy and acetoxy analogues of individual compounds exhibited similar levels of in vivo activity with optimum activity for analogues with Log Ko/w values of 7-8. In contrast, the acetoxy derivatives showed superior in vivo activity against the tobacco whitefly, Bemisia tabaci. Complex III isolated from whitefly was optimally inhibited by hydroxy analogues with lower Log Ko/w values (6.0-6.5) and was also more sensitive than the blowfly enzyme to all the compounds tested.

An insecticidal mixture of tetramethylcyclohexenedione isomers from Kunzea ambigua and Kunzea baxterii.

A mixture of isomers, all 4-[1-(5,7-dihydroxy-6-methyl-4-oxo-2-phenyl-chroman-8-yl)-3-methyl-butyl]-5-hydroxy-2,2,6,6-tetramethyl-cyclohex-4-en-1,3-diones, which comprises a pair of epimers, each of which is a pair of conformers, has been isolated from the hexane extract of the aerial parts of Kunzea ambigua and K. baxterii (Myrtaceae). The mixture exhibits moderate insecticidal activity in comparison with natural pyrethrum extract.