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Synergists are compounds that are either negligibly toxic or non-toxic to insects when applied on their own, but when used in combination with an insecticide, enhance the efficacy of that insecticide. Synergists can be used in combination with pesticides against insects possessing metabolic insecticide resistance mechanisms, since synergists act primarily by inhibiting the metabolic pathway involved in detoxification of an insecticide, and are thus able to temporarily restore a level of susceptibility in the resistant insects (Casida 1970, Metcalf 1967). Synergism would, however, still be found in susceptible insect strains since the detoxification enzymes inhibited by the synergists are still present in baseline amounts. The use of a synergist with pyrethrum thus enhances its efficacy in both resistant and susceptible insect strains, allowing for a more cost-effective formulation of pyrethrum. Processes other than enzyme inhibition, such as increasing the penetration of an insecticide through the cuticle or preventing the deterioration of the insecticide are not considered as classical synergism (Metcalf 1967).

Synergism has particularly been associated with the use of pyrethrum because although pyrethrum is a potent knockdown agent, it is only moderately toxic to most insects without the addition of a synergist. Many insects are able to efficiently detoxify the pyrethrins and thus still survive the application (Casida & Quistad 1995, Scott 1990). A synergist used in combination with pyrethrum inhibits the detoxification of the pyrethrins, and therefore enhances the efficacy of pyrethrum (Casida 1970). For this reason, and due to the high cost of pyrethrum, a search was made for a compound having insecticidal properties to replace pyrethrum, or to use

In the search for a replacement for pyrethrum, it was discovered that sesame oil synergised pyrethrum, having no insecticidal activity on its own. The active compounds were shown to be sesamin, and the more potent, sesamolin (Beroza 1954, Haller et al. 1942a, 1942b). The synergistic effects were attributed to the methylenedioxyphenol (MDP) ring, and the nature of the substituents on the benzene ring was considered important (Haller et al. 1942a). Sesame oil became one of the first commercially available synergists, but its use was limited due to the difficulty in preparing it in suitable quantities, and its low solubility in freon and petroleum hydrocarbons (used in insecticidal sprays). Piperonyl cyclohexenone was subsequently commercialised as a synergist, but an even more active compound was soon found, piperonyl butoxide (Fig. 1.3), which was completely soluble in freon and petroleum hydrocarbons, and relatively non-toxic to mammals (Wachs 1947). Although a number of synergists with the MDP ring have since been commercialised, for example, sulfoxide, propyl isome and Tropital, none have found as much practical use as PBO. PBO is synthesised from the natural product, safrole (Casida 1970). A rich source of safrole originates from trees in Brazil of the genus Ocotea, however, safrole is also produced synthetically (Casida & Quistad 1995).

Fig. 1.3. Structure of piperonyl butoxide (PBO). (Source: Sigma-Aldrich 2010).

The efficacy of the MDP compounds as synergists seems to be influenced by the length of the side chains, the functional groups and the position of the double bond in the side chain attached to the benzene ring (Wen et al. 2006). A long, polyether or oxygen-containing side chain seems to be involved in synergism (Casida 1970). Moore & Hewlett (1958) found that a side chain length of six to ten carbon atoms showed pronounced synergistic activity, whereas fewer or more carbon atoms had less activity. The MDP ring is not a necessity for an effective synergist. A number of synergists without the MDP ring have been commercialised, including SKF 525A, MGK 264 and Synepyrin 500 (Casida 1970).

Chapter One General Introduction _____________________________________________________________________________________________

The action of synergists may not necessarily be restricted to one specific metabolic pathway but can be involved in the inhibition of several enzymes. DEF, for example, acts on both P450s and esterases (Scott 1990). PBO has long been known to inhibit P450s (Wilkinson 1976) but has also been found to inhibit esterases (Gunning et al. 1998, Young et al. 2005, 2006). PBO has also been shown to inhibit AChE (Gunning 2006, Kang et al. 2006). The effects of PBO therefore seem to be multiple, which could explain the high efficacy of PBO.

The efficacy of a synergist is commonly expressed as the synergism factor (SF), which is the ratio of the LD50 of insecticide alone to the LD50 of insecticide with the synergist (Yamamoto 1973). PBO has been shown to increase the toxicity of many insecticides in resistant insects, resulting in high SF values. In B. tabaci, for example, PBO significantly synergised methamidophos, chlorpyrifos, fenvalerate, avermectin, emamectin benzoate, spinosads, fipronil and imidacloprid (Kang et al. 2006). In pyrethroid-resistant H. virescens, PBO mixed with amitraz enhanced the toxicity of cypermethrin (Bagwell & Plapp 1992). Significant synergistic effects were found using PBO with methamidophos, fenvalerate, fipronil and avermectin in P. xylostella,

Phyllotreta striolata (F.) (Coleoptera: Chrysomelidae), Liriomyza sativae Blanchard

(Diptera: Agromyzidae), Propylea japonica Thunberg (Coleoptera: Coccinellidae) and

Cotesia plutellae Kurdjumov (Hymenoptera: Braconidae) (Wu et al. 2007).

There seems to be no one particular best general combination or ratio at which to administer a synergist and an insecticide. The synergistic effect is greatly influenced by the synergist itself, the insecticide used, and the insect species involved. In order to be effective, a synergist should penetrate the insect and be transported to the target enzyme at least equally, but preferably more rapidly, than the insecticide. Once at the target site, the synergist should have a higher affinity for the target enzyme and a lower metabolism rate than the insecticide. The specificity of a synergist can be greatly influenced by any of these processes (Casida 1970, Yamamoto 1973).

A factor known as temporal synergism has also been found to be important when administering synergists in combination with an insecticide. This refers to the amount of time between the application of the synergist and the insecticide (Gunning et al. 1999, Moores et al. 2005, Scott 1990). Pre-treatment with a synergist can increase the amount of synergism found, due to the considerable time it can take for the

(Bingham et al. 2007, Sawicki 1962, Young et al. 2005, 2006). The concept of temporal synergism has led to the development of a novel, microencapsulated insecticide with PBO, where PBO is released initially, followed several hours later by release of the insecticide (Patent: Gunning, R.V & Moores, G.D. Method and

Composition for Combating Pesticide Resistance PCT/GB2003/001861). A

microencapsulated formulation of PBO with alpha-cypermethrin was found to be effective against a few important agricultural insect pest species, H. armigera, M.

persicae, A. gossypii and B. tabaci (Bingham et al. 2007).

With the current focus on decreasing environmental contamination and the increasing demand for organic products, a natural, organically-certifiable compound for use as a synergist would be ideal. PBO, although effective as a synergist, is not classified as an organic product in many countries and an organic compound would therefore be required to replace PBO in formulation with pyrethrum. Natural compounds that have been tested for synergistic activity with pyrethrum include myristicin, safrole, isosafrole, sesamin, sesamolin, piperine, dillapiole, croweacin, elemicin and pongapin (Attri et al. 1973, Brooker et al. unpublished). Searches for effective synergists have not yielded many compounds with potential, however, dill apiole was found to be relatively effective as a synergist (Saxena et al. 1977, Singh

et al. 1976).

An appropriate combination of pyrethrum with a non-toxic, environmentally benign synergist constitutes one of the best and safest means of insect control (Casida 1970) and is the focus of this PhD study. Important qualities for a good synergist would include low mammalian toxicity, effectiveness against a variety of insects, a rapid rate of absorption into the insect body (to reach the target enzyme before the insecticide), stability in storage, good solubility characteristics and a low cost of production or extraction (Beroza & Barthel 1957).