The results of this study highlight which of the pyrethroids used in malaria control are closely related in terms of inhibition of and depletion by P450s. Other studies of structurally diverse pyrethroids have also shown variation in P450 metabolism of pyrethroids with different structure. An in vivo study of the An. funestus strain, FUMOZ-R, which is characterised by upregulated P450 levels without any target site mutations, found that transfluthrin, which contains a polyfluorobenzyl alcohol, was effective in the absence of the generic P450 inhibitor, piperonyl butoxide (PBO), whereas the other pyrethroids that contain common phenoxy benzyl moiety including cypermethrin, ß-cyfluthrin, deltamethrin and permethrin were only effective when partnered with PBO . This effect was associated with an inability of detoxifying enzymes to bind to the uncommon structure of transfluthrin. A similar observation was reported earlier from agriculture where an isogenic metabolic resistance strain isolated from a pyrethroid-resistant field population of Helicoverpa armigera showed significant cross-resistance between the pyrethroids characterised by having both the phenoxy benzyl and aromatic acid moieties whereas the substitution of the phenoxybenzyl group with a polyfluorobenzyl group, as occurs in tefluthrin, benfluthrin and transfluthrin, overcame most of this resistance . These studies support the aim of identifying pyrethroids that are active against resistant populations when P450-mediated resistance plays a major role. In our study, bifenthrin diverged from the other pyrethroids in terms of both inhibition and depletion by P450s, but no susceptibility test data were available for resistance to bifenthrin in populations of African malaria vectors. Susceptibility test data were available for etofenprox and it was found to diverge from the more commonly deployed pyrethroids in terms of inhibition of An. gambiae and An. funestus P450s, and in terms of resistance in An. gambiae s.l. and Ae. aegypti populations.
The susceptibility test data from these populations show strong associations between resistance to the most commonly used pyrethroids (deltamethrin, λ-cyhalothrin, permethrin and α-cypermethrin), in agreement with the results for binding affinity and with earlier studies of spatio-temporal trends in An. gambiae s.l. [3, 35]. The correlations in resistance among these pyrethroids, which were demonstrated in all the major African malaria vectors, suggest that if differences in resistance to these pyrethroids (as well as the less commonly deployed cyfluthrin) are found using susceptibility tests conducted on a small number of field samples of malaria vectors, further evidence should be obtained before any decision is made to switch between them.
Greater differentiation was found for resistance to bifenthrin in terms of both inhibition and depletion by P450s. The results for bifenthrin are interesting because they show that 1) this pyrethroid differs from the other pyrethroids in terms of P450 binding and metabolism, and 2) it may be less susceptible to common P450 enzymes. Bifenthrin is the active ingredient in one indoor residual spray (IRS), Bistar 10WP [2, 49], which is used in India. Bifenthrin IRS was trialled in Nigeria in 2006 and Zambia in 2011 [50–52] but has not been widely deployed in Africa where concerns about the duration of residual activity have been raised [52–54]. There are no field data from susceptibility tests on African malaria vectors conducted using bifenthrin, presumably because this compound is rarely deployed and because there is no recommended diagnostic dose for use in a susceptibility test. One study of Anopheles sinensis in Korea collected blood-fed adults in the field and exposed the F1 larvae to each of the pyrethroids considered by our study. They calculated resistance ratios using LC50 values from a susceptible strain and found that the larvae were most susceptible to bifenthrin, cyfluthrin and etofenprox, in that order, and least susceptible to permethrin . Further evidence comes from studies of Aedes vectors, including three studies that tested bifenthrin . One study in Mexico tested seven populations of Aedes aegypti with eight pyrethroids and compared the concentrations required for 50% knockdown (KC50) and mortality (LC50) to the same values obtained using a susceptible strain to give a resistance ratio (RR) . Across the seven populations, resistance to deltamethrin, lambda-cyhalothrin, permethrin and α-cypermethrin were highly correlated (in terms of both RRKC50 and RRLC50), indicating the existence of strong cross-resistance. However, the resistance values for bifenthrin were not correlated with any those for the other four compounds and the study concluded bifenthrin could be an alternative insecticide for Ae. aegypti in Mexico. Two independent studies in Thailand tested three Ae. aegypti and three Ae. albopictus populations, respectively, and calculated the diagnostic doses for each pyrethroid including bifenthrin using a susceptible strain [57, 58]. In both instances, the population with the highest deltamethrin resistance also had the highest bifenthrin resistance, so no evidence for divergence in resistance was observed for these two species in Thailand. Given the known data noise in susceptibility test results, caution is needed when interpreting the results from a single study at a small number of sites. It is also worth noting that bifenthrin’s relative immunity to depletion by CYP6M2, CYP6P3 and CYP6P9a described here was not found when tested previously . Metabolism assays conducted by two earlier studies showed that CYP6M7, CYP6P9a and CYP6P9b from An. funestus metabolized bifenthrin (62%, 68% and 71% respectively) as well as permethrin, deltamethrin and λ-cyhalothrin (ranging from 46–81% depletion). Field tests for bifenthrin resistance in malaria vector populations are needed before we can reach a firm conclusion about whether bifenthrin should be recommended in situations where resistance to other pyrethroids has been found.
The analyses of binding affinity data and of field data from malaria vector populations both show that resistance to etofenprox diverges, to a degree, from resistance to the more commonly deployed pyrethroids. This result is backed up by data from studies of resistance in Ae. aegypti. However, the depletion activity data suggests that etofenprox is more vulnerable to P450 metabolism and if resistance to this compound is found to be greater in malaria vector populations then a switch would not be advised. A trend for higher resistance to etofenprox was not seen in the data from malaria vector populations but was found in the data from Ae. aegypti populations, although caution is needed when interpreting differences found using susceptibility test data (particularly tests using diagnostic doses that have not been calibrated for Aedes species ). Etofenprox is the active ingredient in two WHO prequalified products; a kit for insecticide-treated nets (Vectron 10EW) and an IRS formulation (Vectron20WP). The latter product is listed by the Global Fund, but etofenprox is not widely deployed in Africa and was last reported as the active ingredient used for IRS in 2012 in parts of Zambia [51, 52].
We found some variation in the relationships among pyrethroids when different types of evidence were considered. In particular, the results for insecticide depletion were largely not repeated in the findings for resistance in mosquito populations. The results for both insecticide inhibition and insecticide depletion depend on which enzymes are included in the activity tests. Seven P450s (three for the depletion analysis) were included here whereas at least 14 have been implicated in An. gambiae s.l. and An. funestus resistance so far [21, 22, 24–32, 46, 60–74] and many more in Aedes vectors . It is also important to note that detoxification by P450s is not the only mechanism of resistance found in these vector species. Target site mutations are common in many of these species [9–13], upregulation of other detoxifying enzymes is also linked to pyrethroid resistance  and there is some evidence for cuticular thickening in resistant mosquitoes . Upregulation of the GSTE2 gene is associated with resistance to both permethrin and deltamethrin, as well as DDT, in An. gambiae and An. coluzzii [71, 73, 77], An. funestus [29, 72, 75] and Ae. aegypti [78–80], and allele frequencies for target site mutations in the voltage-gated sodium channel gene, Vgsc, have been shown to be useful partial predictors of resistance in An. gambiae s.l. . Thus, we would not expect the findings from molecular studies of P450 activity alone to be exactly replicated in field populations, except in instances where P450-mediated metabolic resistance dominates in a mosquito population.
The results for pyrethroid cross-resistance within individual species reported here match our knowledge of other mechanisms of resistance found in these species. Mutations in the Vgsc gene (kdr mutations) confer cross-resistance to pyrethroids and DDT, and are partial predictors of patterns of resistance to these compounds in the An. gambiae complex, but have not been found in An. funestus or other members of the An. funestus subgroup [3, 8–14, 35]. In our study, correlations between pyrethroid and DDT resistance were found for members of the An. gambiae complex but not for the An. funestus subgroup or species. No correlations were found between pyrethroid resistance and resistance to the carbamates or organochlorines, underlining the finding that it is cross-resistance within the pyrethroids, as well as between the pyrethroids and DDT, that is most important. Some metabolic resistance mechanisms do confer cross-class resistance, e.g. between the pyrethroids and DDT and/or the carbamates [24, 30, 32, 74], but the impact of these mechanisms within the array of resistance types that co-occur is more nuanced, and no cross-class resistance other than the aforementioned pyrethroid-DDT resistance in An. gambiae s.l. was detected here.
In conclusion, we have found that evidence for cross-resistance among pyrethroids predicted by SAR studies of metabolic resistance can be detected across African mosquito populations, as exemplified by i) the close associations between the binding affinities of permethrin and deltamethrin to a range of anopheline P450s, ii) the close associations between depletion of permethrin and deltamethrin by these P450s, and iii) correlations in resistance to permethrin and deltamethrin in populations of An. arabiensis, An. coluzzii, An. gambiae and An. funestus. Importantly, populations with higher resistance to one of the pyrethroids studied here, which all contain the common structural motif of phenoxy benzyl alcohol coupled with a cyclopropane ring (the primary target for metabolic oxidation), are likely to have higher resistance to the others and these cross-resistance trends could be detected despite the noise in these susceptibility test data. It is unlikely that resistance to those pyrethroids most commonly deployed for malaria control diverges within vector populations and it would be unwise to switch between these compounds based on the results from a small number of susceptibility tests alone. There are, however, pyrethroids that are not commonly deployed that show greater potential for true divergence in resistance, such as bifenthrin and possibly etofenprox. It is worth noting that there are still significant correlations between resistance to etofenprox and resistance to the pyrethroids in common use, and that this is largely untested for bifenthrin. Systematic SAR analyses of these more structurally diverse pyrethroids are required to estimate the affect of structural diversity on pyrethroid resistance and these findings need to be verified by studies of resistance in wild populations.