In the 1940s, the rise of commercial aviation brought about a steep increase in the risk of malaria and other vector-borne diseases in the US. The insecticide pyrethrum was the preferred method of control, giving a swift ‘knock-down’ and mortality. However, the main producer of pyrethrum was Japan and relationships between the two countries were strained. New research was directed towards either finding a replacement for pyrethrum or enhancing its activity.
Piperonyl butoxide (PBO) offered a partial solution. Mixed with pyrethrum, typically in a ratio of around one part pyrethrum: five parts PBO, the insecticide synergist increased pyrethrum activity by roughly 10-fold, especially when used as a household spray against, for example, houseflies and cockroaches.
First synthesised in 1947, PBO was derived from safrole, a component of sassafras oil from the sassafras tree (Sassafras albidum) – also a flavouring in root beer and the precursor of the drug, Ecstasy. Even today, most cans of household spray will contain either PBO or another synergist, MGK-264 (N-octyl bicycloheptene dicarboximide), in addition to a synthetic pyrethroid insecticide.
The explanation for the pyrethrum enhancement seen with PBO and MGK-264 was originally thought to be because of their inhibition of the P450 enzymes, one of the major insect enzyme detoxification pathways. After penetrating the insect cuticle, insecticides are initially metabolised by P450 and esterase enzymes. Other enzymes called glutathione-S-transferases (GSTs) further breakdown the metabolites of these phase 1 enzymes. By inhibiting the P450 enzymes, the synergists could enhance many classes of insecticides, not just pyrethroids.
Apart from being used in household sprays, synergists today are regularly used as a diagnostic tool to identify resistance mechanisms. The reasoning is clear; if an insecticide alone confers mortality, and this is enhanced by a P450 inhibitor, then it may be deduced that the enzyme was conferring some resistance to that insecticide. Several synergists are described in the literature as having a specific mode of action, ie inhibiting a specific class of enzymes (see table, top of page 29).1
This protocol has been severely undermined since the 1990s, however, with the discovery that synergists do not have a specific action. Thus, it has been reported that PBO inhibits both P450s and esterases, as does MGK-264; 2,3 DEF [S,S,S-tributyl phosphorotrithioate] inhibits P450s as well as esterases; and all these synergists may assist in penetration of the insecticide across the insect cuticle.4,5
Further, it is difficult to assess how much synergist to apply. Generally, researchers will calculate how much synergist alone is required to kill the test insect, and then apply a (just) sub-lethal dose as synergist. But adding so much synergist severely compromises the insect, and precludes any specific action against a single enzyme system. Synergists alone, therefore, cannot be used to identify accurately metabolic resistance.
So have they any role in targeting agricultural insect pests? In Australia, PBO has long been used as a tank mix, often against cotton pests. But although such pests as the cotton bollworm, Helicoverpa armigera, have metabolic resistance mechanisms, it’s now clear that adding PBO into the tank mix is not completely effective even though it inhibits both major phase 1 enzyme systems.
Around 2000, this led Robin Gunning and the author to investigate the time taken for PBO to cross an insect cuticle and inhibit the enzymes. With H. armigera, we learned that the time required was 5 hours, and with the cotton whitefly, Bemisia tabaci, it was even longer at 12 hours.
Such findings led to the idea of ‘Temporal Synergism’, whereby an insect pest is treated by a synergist before exposure to an insecticide several hours later. This would allow time for the synergist to cross the insect cuticle and fully inhibit the metabolic enzymes before insecticide delivery. The sensitised insect now being fully sensitive, or even hypersensitive, would be readily killed by the insecticide.6
The concept was trialled initially against resistant insect pests in the laboratory, where we discovered that a dose of PBO topically applied to an insect several hours before a field dose of insecticide allowed control of even very insecticide-resistant insects. Further, because the metabolic enzymes providing the defence were the same regardless of the insecticide class, such a procedure would enhance control regardless of whether the insecticide was a pyrethroid, carbamate or neonicotinoid.
The major problem now was one of application; even though temporal synergism worked well in the laboratory or greenhouse, spraying twice could prove prohibitively expensive if large areas needed treatment.
The solution was to create a ‘smart’ encapsulation formulation that provided a near-instant burst of PBO upon spraying, followed by a second burst release of insecticide several hours later. This gave the advantage of synergist pre-treatment in a single spray, and formed the basis of the patent submitted by Rothamsted Research UK and the NSW Dept Primary Industries, Australia, in 2002.7
The encapsulated formulation was tested in further trials against resistant pests in 2004/2005, and was found to reduce the amount of pirimicarb required to give 50% mortality by 200-fold against a clone of the green peach aphid (Myzus persicae), and by over 1000-fold against resistant cotton aphid (Aphis gossypii). Similar results were also found for insects resistant to imidacloprid.
In 2011, the author set up ApresLabs on Rothamsted’s new Research and Innovation Campus in Harpenden, UK. In 2013, the author joined other researchers taking part in the two-year EU Framework 7 project, EcoSyn, aimed at devising novel synergists to overcome insecticide resistance and reduce the amounts applied to control insect pests. The project builds on the concept of temporal synergism, and includes partners at Endura (Italy), who are co-ordinating the project; Universita Cattolica del Sacro Cuore (Italy); Rothamsted Research (UK); Babolna Bio (Hungary); Dewar Crop Protection (UK); Ankara Advanced Technologies (Turkey); AgChemAccess (UK); Bee Research Institute (Czech Republic) and ApresLabs (UK).
Novel synergists, initially based on analogues of PBO, are being synthesised by the chemists at Endura. Rothamsted Research has provided recombinant P450 (CYP6CM1 and CYP6CY3) known to be responsible for conferring resistance to important crop pests (Bemisia tabaci and Myzus persicae).
ApresLabs has purified the resistance-associated esterase, FE4, from resistant aphids supplied by Universita Cattolica del Sacro Cuore. In an iterative process, ApresLabs and Endura have studied the interactions of the novel synergists in a Structure-Activity Relationship (SAR) study, modifying the structure of the synergists to provide optimum potency against the metabolic enzymes.
Following the in vitro assays, Universita Cattolica del Sacro Cuore will be largely responsible for in vivo laboratory-based bioassays to ensure the in vitro results can be replicated in a bioassay environment. The most promising candidates will finally be assessed under real field conditions by Dewar Crop Protection and Ankara Advanced Technologies. Babolna Bio is particularly interested in using such products against public health pests, such as cockroaches.
There are two possible obstacles to the widespread acceptance of the technology. These include (1) possible adverse effects to beneficial insects such as bees and other pollinators; and (2) potential selection pressure of the technology to increase the incidence of insecticide resistance, particularly target-site resistance.
To answer the first concern, the Bee Research Institute will be assaying honey bees with the synergists alone and in combination with insecticides. As experts in bee behaviour, the Institute is not only examining bee mortality but also effects on bee learning. To answer the second concern, Rothamsted Research and Universita Cattolica del Sacro Cuore are running long-term selection experiments in which any possible phenotypic and genotypic changes will be identified following insect selection with insecticide alone, compared with insecticide + synergist.
The need for some form of sustained agriculture to supply plentiful food for the world’s growing population – estimated to reach 11bn by 2050 – is well known.
The number of insecticidal actives is decreasing due to new European legislation, while the number of new agrochemical actives coming to market is also reduced because of the prohibitive costs of research and registration. Potent synergists that enhance the activity of insecticides offer a potential solution.
The best-selling insecticides of recent years are the neonicotinoids, most notably imidacloprid. Now resistance has been reported in several cases to this insecticidal class, including both metabolic and target-site insensitivity; however, new research offers some hope.
In the last six months, Universita Cattolica del Sacro Cuore assayed a ‘smart’ neonicotinoid-synergist formulation containing just 50% of the registered field dose of insecticide against resistant strains of the green peach aphid (Myzus persicae) and compared it with a commercial formulation containing the same dose of the same insecticide. The novel formulation killed 100% of the aphids, against just 5% with the commercial formulation.
Myzus persicae is the UK’s most important agricultural pest, as a potent vector of plant viruses it can result in devastation to several arable and horticultural crops throughout Europe, and has evolved resistance to all classes of insecticides used to control it.
These results underline the potential of using synergist; insects that are currently difficult to control with any registered product could be controlled with less than the field rate of existing insecticides.
2 R. Gunning and G. D. Moores, Inhibition of resistance-related esterases by piperonyl butoxide in Helicoverpa armigera (Lepidoptera: Noctuidae) and Aphis gossypii (Hemiptera: Aphididae), pp 215–226. In Piperonyl Butoxide: The Insecticide Synergist, D. G. Jones (ed). London: Academic Press, 1998.
3 N. Sahay and R. A. Agarwal, Chemosphere. 1997, 35, 1011.
4 J. G. Scott, Investigating mechanisms of insecticide resistance: methods, strategies, and pitfalls, pp 39-57. In Pesticide resistance in arthropods, R. T. Roush and B. E. Tabashnik (eds). London, Chapman and Hall, 1990.
5 Y. P. Sun and E. R. Johnson, J.Econ. Entomol., 1972, 65, 349.
6 G. D. Moores, G. Bingham and R. V. Gunning, Outlooks on Pest Management, 2005, 16, 7.
7 Compositions and methods for preventing or reducing resistance of insects to insecticides, patent application number? WO 2003092378 A1
Graham Moores is director of ApresLabs based in Harpenden, UK