Piperonyl Butoxide (PBO) pages

1. PBO - Introduction
2. PBO - Household
3. PBO - Resistance
4. PBO - Dust Mite
5. PBO - Herbicides

3. The use of PBO to control insecticide resistant insect pests.

3.1. Background
The first use of Piperonyl Butoxide, in 1947, was to extend the limited supplies of pyrethrum in the USA; it was several years later that it was found to assist in controlling insecticide resistance. As resistance developed the initial response was to use combinations of insecticides to alleviate the problem. In 1968 Wilkinson reviewed the role of synergists in overcoming problems of resistance and discussed PBO and other methylene dioxyphenol containing compounds. Wilkinson also discussed synergists that are structurally related to the insecticide and compete for the active site of the insect’s detoxifying enzyme thus decreasing the metabolic attack on the insecticide.

Although there was some resistance to the first and second generations of pyrethroid insecticides (see Yamamoto, 1973) it was not until the third generation pyrethroid permethrin that resistance became a serious problem in the control of household and industrial pests.

The 1980s saw significant advances in the understanding of resistance. Liu, Tseng and Sun (1981, see Figure 3.1.) and Glynne Jones (1983) noted that the synergistic effect of PBO was often greatest against resistant insects than against susceptible members of the same species. Raffa and Priester (1985) noted the potential role of synergists in managing insect resistance and stated that ‘synergists are among the most straightforward tools for overcoming metabolic resistance because they can directly inhibit the resistance mechanism itself’. They also demonstrated the important point that when the mortality rate is plotted against dose the effect of a synergist against a resistant insect is to increase the gradient of the regression line. Figure 3.2. shows their results for the action of a synergist on fenvalerate against resistant Spodoptera exigura. It can be seen from the figure that 90% mortality occurs at a much lower dose than non-synergised fenvalerate whereas mortality at 10% occurs at a similar dose. Such an effect is important in understanding the action of a synergist, however, there are many examples of similar studies in which the addition of a synergist results in a steeper regression line but its significance is not discussed by the authors. In a large number of studies comparison of synergised and non-synergised insecticides are made using LD50 data from probit analysis with the result that the effect of the synergist is incorrectly concluded to be minimal.

The experimental methodology of examining the effect of synergists has been found to be critical. It has been found that data obtained from applying space sprays in small chambers such as the Peat Grade apparatus can be very misleading, especially with houseflies. Prior to 1958 very few assays were undertaken using the better method of applying measured drops to individual insects and obtaining sex-differentiated data. From 1956 to 1994 the entomology laboratories of Wellcome and later Roussel Uclaf at Berkhamsted, UK made considerable contributions to the methodology of insecticide and synergist assays; Stewart (1998) and Wickham (1998) summarise this development. An extensive review of insecticide synergists, including wider aspects of synergists usage to control resistance was written by B-Bernard and Philogene (1993).

3.2. Photo-instability
Despite many reports of laboratory studies in which PBO was found to control insecticide resistance, the results of several field studies were poor or indifferent. Indeed, prior to the publication of the PBO monograph (Glynne Jones 1998) it was generally thought in the scientific literature that PBO was relatively stable in light. But the results of the field trials led to the conclusion that the ineffectiveness of PBO was due to its chemical instability in field conditions, in particular that PBO is easily broken down by sunlight. Harbach et al (1998, Chapter 6 of the PBO monograph) gives detailed information on the factors affecting the photostability of PBO. PBO as a film is relatively unstable in sunlight but in the absence of oxygen it remains very stable after months of exposure to strong sunlight. It has also been found in both in vitro and in vivo studies of PBO films on cotton leaves that commercially available UV stabilisers give protection from sunlight for several hours.

3.3. Effect of temperature
There is a general decline in the toxicity of insecticides with an increase in temperature over 20 °C which certainly applies to most pyrethroids. In a study of the effect of different temperatures on the synergising by PBO of the insecticide imidacloprid against adults and larvae of the cat flea, Richman et al (1999) found that the use of PBO would be beneficial at 20, 26 and 30 °C but not at 35 °C which is the probable body temperature of a cat.

There is clearly the need for further study in this area since temperatures in many countries, such as the USA, exceed 40 °C and it is likely that some claims of insect resistance are in fact due to the negative effect of raised temperature on the insecticide and PBO.

3.4. Experimental work on cotton
Very significant amounts of PBO have been used in Australia for grain protection and more recently to overcome resistance in the cotton bollworm. Kennaugh et al (1993) in their study of the effect of PBO on cotton bollworm resistant to permethrin found that there was no evidence for increased permethrin detoxification in the resistant strain. They suggested that the synergism by PBO was not necessarily an indication of resistance due to increased detoxification by cytochrome P450 and that it may indicate an involvement of a cytochrome P450 in the process of penetration of the insecticide through the insect cuticle. Forrester et al (1993) undertook one of the most detailed studies of the action of PBO and other synergists on resistant bollworms.

3.5. Mode of action
It has been generally agreed that PBO works as a synergist because it is a specific inhibitor of mixed function oxidases (MFO). However, this has not explained that the most powerful action of PBO is with carbamate insecticides. In 1991 ICI published an advertising supplement in the September issue of Pest Control in the USA. They reported on a detailed study of nine pyrethroid resistant strains of German cockroach from the USA and found that six strains showed significantly higher levels of one of the main components of the MFO system cytochrome P450 compared to the susceptible strain and they suggested that the addition of PBO blocked this effect. Eight of the resistant strains had high levels of esterase activity which is a primary cause of organophosphate resistance and one strain showed evidence for a nerve insensitivity Kdr-type mechanism. The addition of PBO was thought unlikely to have a beneficial effect in these circumstances.

3.6. Resistance in Cockroaches
Most resistance has been found in various strains of the German cockroach which has displayed a very high level of resistance. The effect of using PBO can be seen in Figure 3.3. which shows PBO added to propoxur in the ratio of 4:1 against resistant (approximately 200-fold) and susceptible male German cockroaches. The results of a KT90 test indicate that with propoxur alone against the susceptible strain the KT90 was after 60–90 minutes and in the resistant strain was after 15 hours whereas with the addition of PBO the KT90 of the susceptible strain was 30 minutes and the KT90 of the resistant strain was 0.7 hours; Figure 3.3. Shows the much steeper regression line obtained with the addition of PBO.

Cochran et al (1987 and 1994) and Scott et al (1990) found that whilst synergists such as PBO had a very important role to play in controlling resistant German cockroaches they would not overcome all pyrethroid resistance problems. Atkinson et al (1991) confirmed this view in their study of the effect of 10 pyrethroid insecticides on a resistant field strain and a susceptible strain of German cockroach. They found that the use of PBO resulted in the partial but significant reduction of resistance and suggested two resistance mechanisms: target site insensitivity and increased metabolism.

Wen and Scott (1997) in their study of the action of imidaclopid against German cockroaches found that after rapid immobilisation some insects recovered 72 hours later. The addition of PBO blocked the recovery and greatly enhanced the 72-hour LD50.

3.7. Resistance in Mosquitoes

3.7.1. The effect of adding PBO to insecticide sprays based on persistent pyrethroids designed to control mosquitoes.
Floore et al (1990) undertook laboratory wind tunnel tests of four permethrin plus PBO formulations compared to a commercial formulation of 4% resmethrin plus 12% PBO against caged adult Culex quinquefasciatus, C. nigripalpus and Aedes taeniorhynchus. The four permethrin formulations differed in the amount of PBO: 0% (92% technical permethrin), 4%, 12% and 20% PBO. The resmethrin plus PBO formulation was used as a positive control for comparisons at 1 hour and 24 hours post-treatment.

They found that the 20% PBO formulation was most effective against C. quinquefasciatus and the 4% formulation the least effective. At 1 hour post-treatment the resmethrin and 20% PBO formulations were equally effective against A. taeniorhyncus and C. quinquefasciatus, but at 24-hours post-treatment the 20% PBO formulation was 1.3 times more effective on both species than the resmethrin formulation. They also found that the resmethrin formulation was 1.2 times more effective then the 20% PBO formulation against C. nigripalpus 1 hour post-treatment but at 24-hour post-treatment the 20% PBO formulation was >1,6 times as effective than the resmethrin formulation. The 92% technical permethrin was not evaluated at 1-hour post-treatment and was the least effective of all formulations tested.

Thomas et al (1991) examined the use of PBO against resistance to deltamethrin in C. quinquefasciatus. They undertook larval selection for 40 generations which resulted in a 1449-fold increase in resistance to deltamethrin. When the larvae were subjected to selection with deltamethrin and PBO (in ratio of 1:5) the speed of selection for deltamethrin resistance was slowed by 17-63%. In a parallel study a strain showing 137-fold resistance to deltamethrin was subjected to selection with synergised deltamethrin and showed a 76% reversion of deltamethrin resistance in the first generation and subsequent development of deltamethrin resistance was retarded. The data indicate the efficacy of PBO in enhancing the usefulness of deltamethrin. Selection for deltamethrin resistance also resulted in cross-resistance to DDT suggesting the probable involvement of a Kdr gene.

Kumar et al (1991) investigated the role of mono-oxygenases as a mechanism of resistance to deltamethrin in larvae of Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi. Strains of all three species were produced in laboratory selection using deltamethrin, DDT or deltamethrin plus PBO (1:5). There was a significant correlation between mono-oxygenase activity (MOA) and larval LC50 to deltamethrin in various strains of all three species. In addition, the activity of glucose-6-phosphate dehydrogenase (G6PD), the main NADPH-generating enzyme for MOA, also showed enhanced activity in deltamethrin and DDT-selected strains. The results suggest that deltamethrin resistance in the larvae of all three species is mainly due to the detoxification of deltamethrin by microsomal mono-oxygenases. They found that high activity of G6PD observed in DDT-selected strains appears to be related to its role as a rate-limiting enzyme in GSH-dependant dehydrochlorination of DDT.

Floore et al (1992) evaluated three cypermethrin plus PBO (1:1, 1:5 and 1:10) formulations using a 4% resmethrin plus 12% PBO formulation as a positive control in a wind tunnel test using laboratory reared susceptible Culex quinquefasciatus. Based on the LC90 data the three cypermethrin plus PBO formulations were 2.6–3.7 times more toxic than the resmethrin plus PBO formulation at 24 our post treatment.

Rodriguez et al (1995) examined the mechanisms of resistance in Culex quinquefasciatus from six municipalities in Havana City, Cuba in order to determine their genetic frequency. Mosquitoes were collected in February to March 1993 and maintained in the laboratory for four generations. Increased esterases followed by altered acetylcholinesterase were the major resistance mechanisms in Havana. Resistance to chlorpyrifos was found for the first time, as well as continued resistance to malathion and propoxur; a deterioration in susceptibility to pyrethroids was detected.

The synergised effect of S,S,S-tributylphosphorotrithioate (DEF) and PBO was examined and it was found that esterases and mixed function oxidases were involved in the resistance to the pyrethroids. Using polyacrylamide gel electrophoresis it was found that the combination of esterases probably associated with pyrethroid resistance was B1-A6-B6 which appeared with the greatest percentage. Values of genetic frequency in the six municipalities were found to be high for both esterase and acetylcholinesterase genes.

3.7.2. Ratio of pyrethroid to PBO and particle size for mosquito control
For household formulations of insecticides directed against houseflies the user expects a high rate of knock down (KD). This property is, however, of much less importance for formulations designed to kill mosquitoes. Successful control of mosquitoes is usually measured in terms of the cessation of biting by female mosquitoes; a paralysed mosquito may fall to the ground unnoticed. It has also been found that a lower ratio of synergist to pyrethroid (1:4) is required for killing mosquitoes than the high ratio required for killing flies (1:5 upwards). For rapid KD of houseflies, spray particles above a diameter of 60 micrometers are optimum whereas for adult mosquitoes particles ranging from 12 to 40 micrometers give best results.

3.8. Resistance in Houseflies

3.8.1. The control of resistant houseflies
In many of the warmer countries of Europe the available household insecticides are not now effectively controlling female houseflies which have developed into a serious nuisance. In Spain, for example, the supermarkets sell 750ml aerosol cans of water-based insecticide containing borderline amounts of actives. The level of active gives over 90% control of male flies but often less than 20% control of female flies; gravid females may require over 3.2 times more active than males to effect kill. The market is very competitive and the supermarkets insist on formulators absorbing price increases.

One company in Spain selling technical products has been actively discouraging the use of PBO in household formulations, presumably to promote their own products and in many areas of the country no formulations containing PBO are on sale. A recent attempt to control resistant flies and mosquitoes has been the introduction of smaller, ‘concentrate’ cans for example, a 200ml can containing cyfluthrin 0.05% and transfluthrin 0.08% with smaller particle size which the manufacturers claim is equivalent to a 400ml can. Another product contains neopynamin forte 0.33% and sumithrin 0.132% and is labelled ‘insecticida concentrado – De Triple efficacia’, it contains no PBO even though performance against female flies is likely to be limited and the mortality/dosage regression line of low gradient.

Formulations with steeper regression lines are more likely to be regarded as highly effective by the end user. It has been estimated that in over 85% of cases the addition of PBO will increase the gradient of the regression line which is easily calculated using the appropriate computer program.

It is, however, important to compare the effectiveness of adding PBO not only at the LD50 point but at the LD90 point as well.

Before a new product formulation is approved it is recommended that tests are undertaken using half the proposed concentration of the actives as this reduces the chances of high total mortality over 90% and enables better comparisons with other formulations. The testing should use the measured drop technique on both male and female flies separately for both resistant and susceptible strains.

3.8.2. The effect of PBO on housefly resistance
Macdonald et al (1983) selected a strain of houseflies with a 73-fold resistance to permethrin. They found that a 10:1 mixture of PBO and permethrin increased permethrin susceptibility 5-fold and reduced heterogenicity in the resistant permethrin selected strain. De Vries and Georghiou (1981) studied houseflies resistant to permethrin and found a 12-fold synergism with addition of PBO and subsequently related resistance to a decreased rate of insecticide penetration and that PBO acted by increasing the rate of penetration. However, Bull and Pryor (1990) studied the in vivo and in vitro fate of fenvalerate in pyrethroid-resistant and susceptible flies. They found that co-administered PBO slowed the rate at which fenvalerate penetrated the cuticle of resistant flies and that PBO significantly reduced the metabolic degradation of fenvalerate. Clearly further work is required on effect of PBO on insect cuticles. More recently Scott and Wen (1997) found the new insecticide fipronil highly toxic to susceptible and resistant strains of houseflies and that the addition of PBO increased the toxicity 10-fold.

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