A sublethal dose of thiamethoxam causes a reduction in xylem feeding by the bird cherry-oat aphid (Rhopalosiphum padi), which is associated with dehydration and reduced performance.

The active ingestion of xylem sap by aphids is hypothesised to be an important mechanism for rehydration. When starved bird cherry-oat aphids (Rhopalosiphum padi) were allowed to feed on wheat (Triticum aestivum) treated with a sublethal dose of the xylem-mobile neonicotinoid thiamethoxam, analysis of feeding behaviours using the electrical penetration graph revealed a reduction in xylem feeding that was reversed on removal of the toxin. To test the importance of xylem-feeding behaviour as a rehydration mechanism, the effects of the sublethal dose of thiamethoxam on aphid water content, honeydew excretion, growth and fecundity were investigated. Body water contents of starved R. padi feeding on wheat treated with thiamethoxam were significantly reduced compared to aphids feeding on wheat treated with distilled water (74.5+/-0.23 and 75.6+/-0.18%, respectively). In addition, the sublethal dose of thiamethoxam had detrimental effects on aphid performance. At reproductive maturity, aphids that had been born on wheat treated with thiamethoxam were significantly smaller (as measured by body plan area; 1.07+/-0.03mm(2)), lighter (0.31+/-0.04mg) and less fecund (2.85+/-0.36nymphs/day) than aphids born on wheat treated with distilled water (1.87+/-0.02mm(2), 0.72+/-0.03mg, 11.28+/-0.58nymphs/day, respectively). Regardless of whether the observed impairment of xylem feeding is due to a neurotoxic or an antifeedant effect, these results have important implications for commercial crop protection as the behaviour-modifying effects of the sublethal dose of thiamethoxam may change the efficacy of this pesticide throughout the growing season.

[3H]-Methyllycaconitine: a high affinity radioligand that labels invertebrate nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChR) of insect and other invertebrates are heterogeneous and new tools are needed to dissect their multiplicity. [(3)H]-Methyllycaconitine ([(3)H]-MLA) is a novel radioligand which is a potent antagonist at vertebrate alpha7-type nAChR. Putative invertebrate nAChR of the aphid Myzus persicae, the moths Heliothis virescens and Manduca sexta, the fly Lucilia sericata, and the squid Loligo vulgaris were investigated in radioligand binding studies with [(3)H]-MLA. Saturable binding was consistent with a single class of high affinity binding sites for each of these invertebrates, characterised by a dissociation constant, K(d), of approximately 1 nM and maximal binding capacities, B(max), between 749 and 1689 fmol/mg protein for the insects and 14,111 fmol/mg protein for squid. [(3)H]-MLA binding to M. persicae membranes was characterised in more detail. Kinetic analysis demonstrated rapid association in a biphasic manner and slow, monophasic dissociation. Displacement studies demonstrate the nicotinic character of [(3)H]-MLA binding sites. Data for all nicotinic ligands, except MLA itself, are consistent with displacement from a high and a low affinity site, indicating that displacement is occurring from two or more classes of nicotinic binding site that are not distinguished by MLA itself. Autoradiographic analysis of the distribution of [(3)H]-MLA binding sites in Manduca sexta shows discrete labelling of neuropil areas of the optic and antennal lobes.

Characterization of a nicotinic acetylcholine receptor from the insect Manduca sexta.

Manduca sexta is a nicotine-insensitive insect, the larval form of which feeds on tobacco. It has been postulated that its nicotine insensitivity may reflect the presence of a modified nicotinic acetylcholine receptor whose alpha subunits lack the amino acid residues necessary for binding nicotine: we have performed ligand binding assays and molecular cloning to examine this hypothesis. [125I]alpha-bungarotoxin bound specifically to both larval and adult membranes, with Kd values of 7.6 and 6.5 nM and Bmax values of 119 and 815 fmol/mg protein, respectively. The pharmacological profile of [1251]alpha-bungarotoxin binding was similar in both tissues. In particular, nicotine (Ki values: 1.6 microM and 2 microM for larvae and adults, respectively) competed with an affinity similar to that found for nicotine-sensitive insects. No alpha-bungarotoxin-insensitive binding sites labelled by [3H]epibatidine could be detected. Using the alpha-like subunit from the locust Schistocerca gregaria to probe two cDNA libraries, and by inverse PCR on circularized genomic DNA from Manduca sexta, we have obtained overlapping cDNA clones that contain the complete coding sequence of a putative nicotinic subunit from Manduca sexta (MARA1). No other alpha-subunit cDNAs were isolated using this probe, although it hybridized to multiple bands on Southern blots. The sequence of MARA1 is consistent with an alpha-like subunit capable of binding alpha-bungarotoxin, and it retains all those amino acids implicated in nicotine binding to vertebrate nicotinic receptors. Taken together, these findings provide no support for the hypothesis that the nicotine insensitivity of Manduca sexta is the result of a nicotinic receptor with diminished nicotine binding.