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Evaluation of the pyrrole insecticide chlorfenapyr against pyrethroid resistant and susceptible Anopheles funestus (Diptera: Culicidae)

2009; Wiley; Linguagem: Inglês

10.1111/j.1365-3156.2009.02416.x

1365-3156

Shüné V. Oliver, Maria L. Kaiser, Oliver R. Wood, Maureen Coetzee, M Rowland, Basil D. Brooke,

Insect Resistance and Genetics

Objective To evaluate the pyrrole insecticide chlorfenapyr, which has a novel non-neurotoxic mode of action and is a promising alternative to conventional adulticides, against Anopheles funestus. Method The toxicity of a range of concentrations of chlorfenapyr against pyrethroid resistant and susceptible laboratory reared southern African An. funestus was assessed using standard WHO protocols and analysed using probit analysis. Results The pyrethroid resistant strain showed consistently higher LD50 and LD95 values compared to the susceptible strain, but these differences were not statistically significant and the magnitude was twofold at most. The LD50 values recorded for An. funestus are approximately three-fold higher than those reported elsewhere for other species of anopheline. Conclusions Monooxygenase based pyrethroid resistance in An. funestus does not influence the toxic effect of chlorfenapyr. It is unlikely that such a small decrease in susceptibility of An. funestus to chlorfenapyr relative to other anophelines would have any operational implications. Chlorfenapyr is an important addition to insecticides available for malaria vector control, and could be used as a resistance management tool to either circumvent or slow the development of resistance. Objectif: Evaluer le chlorofénapyr, insecticide pyrrole, qui possède un nouveau mécanisme d'action neurotoxique et est une alternative prometteuse aux adulticides classiques, contre Anopheles funestus. Méthode: La toxicité d'une plage de concentrations de chlorofénapyr contre des souches d'An. funestus d'Afrique australe élevées au laboratoire, résistantes et sensibles pyréthroïdes, a étéévaluée en utilisant des protocoles standards de l'OMS et analysées en utilisant l'analyse probit. Résultats: La souche résistante aux pyréthroïdes a montré des valeurs de DL50 et LD95 toujours plus élevée comparéà la souche sensible, mais ces différences n'étaient pas statistiquement significatives et la magnitude était de deux fois au maximum. Les valeurs de DL50 enregistrées pour A. funestus sont environ trois fois plus élevées que celles rapportées ailleurs pour d'autres espèces d'anophèles. Conclusions: La résistance de An. funestus aux pyréthroïdes basée sur la monooxygenase n'influence pas l'effet toxique du chlorofénapyr. Il est peu probable que cette légère diminution de la sensibilité d'An. funestus au chlorofénapyr comparéà d'autres anophèles ait des implications opérationnelles. Le chlorfenapyr est un important insecticide additionnel pour la lutte contre le vecteur de la malaria et pourrait être utilisé comme un outil dans la gestion de la résistance pour contourner ou ralentir le développement de la résistance. Objetivo: Evaluar el insecticida pirrol clorfenapyr, el cual tiene un modelo de acción novedoso, no-neurotóxico, y es una alternativa prometedora a los adulticidas frente a Anopheles funestus. Método: La toxicidad de una rango de concentraciones de clorfenapyr frente a cepas de laboratorio, del sur de África, de An. funestus susceptibles y resistentes a piretroides se evaluó utilizando protocolos estándar de la OMS y se analizó utilizando la metodología probit. Resultados: Las cepas resistentes a piretroides mostraron de forma consistente valores LD50 y LD95 más altos que la cepa susceptible, pero estas diferencias no eran estadísticamente significativas y la magnitud era como mucho del doble. Los valores LD50 para An. funestus eran aproximadamente tres veces más que aquellos reportados en otros lugares para otras especies anofelinas. Conclusiones: La resistencia a piretroides basada en monooxigenasa en An. funestus no tiene influencia en el efecto tóxico del clorfenapyr. Es poco probable que una disminución tan pequeña en la susceptibilidad de An. funestus al clorfenapyr con relación a otros anofelinos tenga implicaciones operativas. El clorfenapyr es una adición importante a los insecticidas ya disponibles para el control de los vectores de malaria, y podría utilizarse como una herramienta para el manejo de resistencias, bien para sortear o para retrasar el desarrollo de resistencias. Anopheles funestus Giles is a major malaria vector species in the Afrotropical region. It is the nominal member of a species group that comprises several subspecies of which only An. funestus is implicated as a major malaria vector (Gillies & De Meillon 1968; Harbach 2004). Anopheles funestus populations have shown resistance to DDT, dieldrin, malathion and fenitrothion in Mali; to DDT, carbamate and pyrethroid in Ghana; to dieldrin in Benin, Cameroon, Ghana, Nigeria and Kenya (Brown 1986; Coetzee et al. 2006; Okoye et al. 2008) and to pyrethroid and carbamate in South Africa and Mozambique (Hargreaves et al. 2000; Brooke et al. 2001; Casimiro et al. 2006). Pyrethroid resistance in An. funestus was first detected in 1999 in southern Africa (Hargreaves et al. 2000). Resistance to deltamethrin, possibly confounded by anti-malarial drug resistance, resulted in a malaria epidemic in South Africa where malaria incidence rose from 8750 to 64 622 cases per year between 1996 and 2000 (Coetzee 2005). Re-introduction of DDT for vector control and a change in the prescribed anti-malarial drug regimen led to a marked reduction in malaria cases in South Africa with only 5210 cases reported in 2007 (unpublished Department of Health statistics). Since the introduction of pyrethroids for vector control, no new malaria vector adulticides have been approved by the World Health Organization (Nauen 2007). Chlorfenapyr is a novel insecticide used for the control of veterinary and agricultural pests (Lovell et al. 1990). As a pro-insecticide activated by oxygenases to a more toxic metabolite AC-303,268, a powerful mitochondrial uncoupler (Black et al. 1994), it has a unique mechanism of action distinct from the commonly used neurotoxic insecticides. Because it is activated by cytochrome P450s, the monooxygenase inhibitor piperonyl butoxide (PBO) antagonises its activity (Black et al. 1994). It has WHO toxicological classification III, and like many insecticides is classified as 'slightly hazardous' (Tomlin 2000). Chlorfenapyr is used to control two-spotted spider mites Tetranychus urticae (Herron & Rophail 2003) and subterranean termites Reticulitermes hesperus (Rust & Saran 2006). It has also been used in the cattle industry in impregnated ear tags to control Haematoba irritans (Guglielmone et al. 2000). Chlorfenapyr is an effective larvicide against the dengue vector Aedes aegypti, and moderately toxic to Ae. aegypti adults (Paulet al. 2006). Chlorfenapyr was included in a survey of 19 insecticides, with a variety of modes of action, tested for toxicity to Ae. aegypti, Culex quinquefasciatus and An. quadrimaculatus. Of these three species, C. quinquefasciatus was least susceptible, while An. quadrimaculatus was most susceptible (Pridgeon et al. 2008). The toxicity of chlorfenapyr to An. gambiae and An. stephensi has been evaluated and shows no reduction in susceptibility in mosquitoes carrying knockdown resistance (kdr) or insensitive acetylcholinesterase (Ace-1R) mutations (N'Guessan et al. 2007). It has been identified as a potential adjunct to pyrethroids in ITN treatment following promising results in field trials in Tanzania and Benin which were designed to assess its efficacy against An. arabiensis, An. gambiae and C. quinquefasciatus populations (Mosha et al. 2008; N'Guessan et al. 2009). In this study, we present the first evaluation of chlorfenapyr against laboratory pyrethroid resistant and susceptible strains of An. funestus from southern Africa. Two laboratory colonies of An. funestus were used: (i) FANG, an insecticide susceptible colony originating from southern Angola and colonised in January 2003; (ii) FUMOZ-R, a colony selected for pyrethroid resistance (Hunt et al. 2005) originating from southern Mozambique, and colonized in 2001. Resistance to pyrethroids in FUMOZ-R is primarily based on the detoxifying properties of elevated P450 monooxygenases (Brooke et al. 2001; Amenya et al. 2008; Wondji et al. 2009). These colonies are housed at the National Institute for Communicable Diseases/NHLS in Johannesburg and are reared under standard insectary conditions (Hunt et al. 2005). The following range of chlorfenapyr concentrations was assessed against FUMOZ-R and FANG: 4%; 2%; 1%; 0.5%; 0.25%; 0.125% and 0.0625%. Standard WHO insecticide susceptibility test kits were lined with filter papers impregnated with the above concentrations of chlorfenapyr. Each test paper was treated with a 2 ml solution of chlorfenapyr dissolved in olive oil and acetone to the required concentration. Susceptibility tests included seven replicates per chlorfenapyr concentration per colony with ∼20 non-blood-fed female mosquitoes (2–3 days old) per exposure tube. Exposures lasted 1 h. Controls included exposures to filter papers treated with solvents (acetone and olive oil) only. Mortality was recorded 24, 48 and 72 h post-exposure. All mosquitoes were provided with a 10% sugar solution for the entire post-exposure period. The results of replicates where control mortality exceeded 10% were discarded. Used insecticide treated papers were replaced with fresh papers weekly. All data analysis was performed using statistix 7 (Analytical Software, Tallahassee, FL, USA). Lethal Dose (LD)50s, LD95s and resistance ratios were determined using regression analysis of logarithmically transformed mortality data. Analysis of variance (anova) was used to compare LD50s and LD95s between FANG and FUMOZ-R 24 h, 48 h and 72 h post-exposure to chlorfenapyr. Mean knockdown immediately after 1 h exposure to 4% chlorfenapyr was 19.2% (SE 9.9) for FANG and 1.1% (SE 0.7) for FUMOZ-R. This apparent difference is not mirrored statistically based on a two-sample t test (P = 0.1). There was either no or negligible knockdown immediately after 1 h exposure for all other concentrations tested against both An. funestus strains. The dose-mortality charts shown in 1-3 suggest that FUMOZ-R was less susceptible to chlorfenapyr exposure than FANG, a trend consistent at 24, 48 and 72 h post-exposure. Further, FUMOZ-R showed higher mean LD50 and LD95 levels than FANG through all post-exposure periods (Table 1). Resistance ratios (RR) were estimated using the LD50 and LD95 means of FUMOZ-R and FANG and are also given in Table 1. Resistance ratios are consistently greater than 1, reinforcing the apparent difference in response to chlorfenapyr exposure between strains. However, anova showed that the differences in lethal dose between the two strains at LD50 and LD95 were not significant for any of the post-exposure periods (Table 1). Mean percentage mortalities for An. funestus FANG (insecticide susceptible) and FUMOZ-R (pyrethroid resistant) 24 h post-exposure to chlorfenapyr. Mean percentage mortalities for An. funestus FANG (insecticide susceptible) and FUMOZ-R (pyrethroid resistant) 48 h post-exposure to chlorfenapyr. Mean percentage mortalities for An. funestus FANG (insecticide susceptible) and FUMOZ-R (pyrethroid resistant) 72 h post-exposure to chlorfenapyr. The introduction of new insecticides formulated for public health has not kept pace with the rate of insecticide resistance development in mosquito vectors. The requirement for new pesticides, whether chemical or biological, to replace or supplement currently used compounds remains pressing (Zaim & Guillet 2002). Chlorfenapyr presents one such novel vector control insecticide. Although both pyrethroid resistant and susceptible An. funestus cohorts succumbed to chlorfenapyr exposure, their responses appeared to be different. The resistant FUMOZ-R strain showed consistently higher LD50 and LD95 values than the susceptible FANG strain. Although this difference is reflected in the resistance ratios, it is not statistically significant based on anova of LD50 and LD95 values. The numerical differences recorded are likely due to variation normally associated with biological repeats of insecticide susceptibility tests, the net effect of physiological differences between the two colonies which do not share the same genetic background. Based on the exposure data described here, An. funestus showed a slightly higher tolerance to chlorfenapyr than did An. stephensi and An. gambiae. In comparison with N'Guessan et al. (2007), FANG showed a 3.2-fold increase in LD50 over susceptible An. stephensi, and FUMOZ-R showed a 2.81-fold increase in LD50 over An. stephensi resistant to organophosphates and pyrethroids. It is unclear as to whether the decreased susceptibility to chlorfenapyr recorded for An. funestus will translate into an operational problem or whether the apparent difference with An. gambiae and An. stephensi is caused by anything more than differences in laboratory testing conditions rather than species differences. There is no clear relationship between dosages used for laboratory bioassays and operational dosages applied in field settings because mosquito contact times between bioassay and field applications vary considerably, and so bioassay results are not necessarily predictive of field impact. Chlorfenapyr is a pro-insecticide activated by the oxygenase function of cytochrome P450s (Black et al. 1994). Negative cross-resistance, i.e. an increased susceptibility to chlorfenapyr in strains with P450-mediated insecticide resistance, might be expected, as was observed in cattle horn flies, Haematobia irritans (Sheppard & Joyce 1998) and tobacco budworms, Heliothis virescens (Pimprale et al. 1997). However, this effect tends not to appear in mosquitoes. For example, although pyrethroid resistance in the Dub234 An. stephensi strain is conferred by elevated P450 activity as well as kdr, no negative cross resistance to chlorfenapyr was observed (N'Guessan et al. 2007). Further, it was noted that synergism of monooxygenase activity by the P450 inhibitor piperonyl butoxide (PBO) did not significantly alter the toxicity of chlorfenapyr to susceptible fourth instar Ae. aegypti larvae (Paul et al. 2006). Pyrethroid resistance in the An. funestus FUMOZ-R strain used in this study is based on duplication and over-expression of the CyP6P4 and Cyp6P9 monooxygenase genes (Amenya et al. 2008; Wondji et al. 2009). The data presented here suggests that an increased expression level of these particular genes is neither associated with positive nor negative cross resistance to chlorfenapyr. By comparison to most other insecticides used for public health, chlorfenapyr is slow acting, which is not necessarily a limiting factor for IRS treatment since many once successful IRS treatments were slow acting (e.g. with cyclodiene insecticides in the 1950s). While the capacity to develop resistance to chlorfenapyr has been demonstrated in other insect species such as the two-spotted spider mite (Herron & Rophail 2003) and the spotted bollworm Earias vitella (Ahmad & Iqbal Arif 2009) this novel insecticide should prove to be an important addition to malaria vector control agents and could be used as a resistance management tool to either circumvent or slow the development of resistance in target vector populations. This project was supported by the Innovative Vector Control Consortium and the NRF/DST Research Chair award to MC.