Richards, David
(2012)
Investigating ladybird (Coleoptera : Coccinellidae) alkaloids as novel sources for insecticides: differential inhibition of the vertebrate and invertebrate nicotinic acetylcholine receptor using harlequin ladybird (Harmonia axyridis) extract and synthetic hippodamine.
MRes thesis, University of Nottingham.
Abstract
Ladybird beetles (Coleoptera:Coccinellidae) are ubiquitous, being found on every continent with the exception of Antarctica, and inhabiting a huge range of habitats. Roughly 6000 sp. have been described, comprising at least 490 genera. When disturbed, ladybirds ‘reflex bleed’ (autohemorrhage) from the femoro-tibial joints, producing a viscous, yellow liquid which has a very distinctive odour, this has been shown to be an effective deterrent against predation from both birds and arthropods. Feeding trials have demonstrated that many ladybirds are considered unpalatable and unacceptable as food sources by many potential predators. The bright colouration displayed by many coccinellids is thought to function as an aposematic warning. This has been demonstrated experimentally using Japanese quails, where colour was shown to be a more effective feeding deterrent than either taste or smell. A number of novel compounds have been isolated from this fluid, including alkaloids. It has been hypothesised that alkaloids within the defensive fluid will serve a toxic function, and this is the basis of the aposematic colouration.
The aim of this work was to investigate the toxinological properties of alkaloid extracts of the harlequin ladybird (Harmonia axyridis) as well as a synthetic analogue of hippodamine, an alkaloid isolated from the convergent ladybird (Hippodamia convergens). We wished to establish whether these alkaloids display activity against nicotinic acetylcholine receptors (nAChR), and if so, whether they show any selectivity towards vertebrate or invertebrate nAChR. To do this we collected harlequin ladybirds and extracted the alkaloids using methanol, followed by acid-base extraction. HPLC revealed a number of compounds with elution times consistent with alkaloids. To test for nAChR activity we employed whole-cell patch clamp combined with perfusion of human muscle cells (TE671) and locust (Schistocerca gregaria) neurons. Both H. axyridis extract and hippodamine were found to be potent antagonists to nAChR in both TE671 cells and locust neurons. Furthermore both were found to be considerably more potent to locust nAChR than TE671. H. axyridis extract had IC50 values of 2.12 μg/ml and 39.9 ng/ml in TE671 and locust neurons respectively with a holding potential of -50mV. Hippodamine had IC50 values of 22.3 μM and 18.5nM in TE671 and locust neurons respectively with a holding potential of -50mV. F-test comparisons revealed that both test compounds were significantly more potent to locust neurons than to TE671 cells (H. axyridis p=0.0015, hippodamine p=0.0001) at holding potentials of -50mV and -100mV. F-test comparisons between holding potentials revealed a voltage dependence in hippodamine when applied to locust neurons (p=0.0153), but no other voltage dependent relationships were identified.
The results of these experiments reveal that ladybird alkaloids do display toxicity, potently antagonising the nAChR. Further to this, both of those tested displayed selective toxicity towards locust nAChR of between 41 and 1205 fold potency for H. axyridis extract and hippodamine respectively. It remains to be seen how this is achieved, but this high selectivity warrants further investigation into the use of these alkaloids as lead structures for insecticides.
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