Major effect genes or loose confederations? The development of insecticide resistance in the malaria vector Anopheles gambiae

Insecticide use in public health and agriculture presents a dramatic adaptive challenge to target and non-target insect populations. The rapid development of genetically modulated resistance to insecticides is postulated to develop in two distinct ways: By selection for single major effect genes or by selection for loose confederations in which several factors, not normally associated with each other, inadvertently combine their effects to produce resistance phenotypes. Insecticide resistance is a common occurrence and has been intensively studied in the major malaria vector Anopheles gambiae, providing a useful model for examining how insecticide resistance develops and what pleiotropic effects are likely to emerge as a consequence of resistance. As malaria vector control becomes increasingly reliant on successfully managing insecticide resistance, the characterisation of resistance mechanisms and their pleiotropic effects becomes increasingly important

The effect of a single blood meal on the phenotypic expression of insecticide resistance in the major malaria vector Anopheles funestus

<p>Abstract</p> <p>Background</p> <p><it>Anopheles funestus </it>is a major malaria vector in southern Africa. Vector control relies on the use of insecticide chemicals to significantly reduce the number of malaria vectors by targeting that portion of the female population that takes blood meals and subsequently rests indoors. It has been suggested that the intake of a blood meal may assist female mosquitoes to tolerate higher doses of insecticide through vigour tolerance. It is hypothesized that during the process of blood digestion, detoxification mechanisms required for the neutralizing of harmful components in the blood meal may also confer an increased ability to tolerate insecticide intoxication through increased enzyme regulation.</p> <p>Methods</p> <p>Bottle bioassays using a range of concentrations of the pyrethroid insecticide permethrin were performed on pyrethroid susceptible and resistant laboratory strains of <it>An. funestus </it>in order to detect differences in insecticide susceptibility following a single blood meal. Based on these results, a discriminating dosage was identified (double the lowest dosage that resulted in 100% mortality of the susceptible strain). Blood-fed and unfed females drawn from the resistant strain of <it>An. funestus </it>were then assayed against this discriminating dose, and the percentage mortality for each sample was scored and compared.</p> <p>Results</p> <p>In the insecticide dose response assays neither the fully susceptible nor the resistant strain of <it>An. funestus </it>showed any significant difference in insecticide susceptibility following a blood meal, regardless of the stage of blood meal digestion. A significant increase in the level of resistance was however detected in the resistant <it>An. funestus </it>strain following a single blood meal, based on exposure to a discriminating dose of permethrin.</p> <p>Conclusion</p> <p>The fully susceptible <it>An. funestus </it>strain did not show any significant alteration in susceptibility to insecticide following a blood meal suggesting that vigour tolerance through increased body mass (and increased dilution of internalized insecticide) does not play a significant role in tolerance to insecticide intoxication. The increase in insecticide tolerance in the pyrethroid resistant strain of <it>An. funestus </it>following a blood meal suggests that insecticide detoxification mechanisms involved in insecticide resistance are stimulated by the presence of a blood meal prior to insecticide exposure, leading to enhanced expression of the resistance phenotype. This finding may be significant in terms of the methods used to control indoor resting populations of <it>An. funestus </it>if the mass killing effect of insecticide application proves increasingly inadequate against blood-feeding females already carrying the insecticide resistance phenotype.</p

Insecticide resistance in Anopheles arabiensis (Diptera: Culicidae) from villages in central, northern and south west Ethiopia and detection of kdr mutation

<p>Abstract</p> <p>Background</p> <p><it>Anopheles arabiensis </it>is the major vector of malaria in Ethiopia. Malaria vector control in Ethiopia is based on selective indoor residual spraying using DDT, distribution of long lasting insecticide treated nets and environmental management of larval breeding habitats. DDT and pyrethroid insecticides are neurotoxins and have a similar mode of action on the sodium ion channel of insects. It was therefore necessary to verify the insecticide susceptibility status of <it>An. arabiensis</it>, to better understand the status of cross-resistance between DDT and the pyrethroids in this species as well as to detect a resistant gene.</p> <p>Methods</p> <p>Standard WHO insecticide susceptibility tests were conducted on adults reared from larval and pupal collections from breeding sites at three villages namely: Sodere in the Rift Valley, Gorgora in the north and Ghibe River Valley in the south west of Ethiopia. The occurrence of cross-resistance between pyrethroids and DDT was determined using a DDT selected laboratory colony originally collected from Gorgora. Phenotypically characterized mosquitoes were tested for the presence of knockdown resistance (<it>kdr</it>) alleles using the standard polymerase chain reaction assay.</p> <p>Results</p> <p>All <it>An. gambiae </it>s.l. specimens assayed by PCR were identified as <it>An. arabiensis</it>. The knockdown and mortality results showed <it>An. arabiensis </it>resistance to DDT in all villages, resistance to deltamethrin and permethrin in the Ghibe River Valley and permethrin resistance in Gorgora. Bioassay susceptibility tests also indicated the presence of cross-resistance between DDT and permethrin, but not between DDT and deltamethrin. The knockdown resistance <it>(kdr) </it>mutation of leucine to phenylalanine in the sodium ion channel gene was detected in populations from Gorgora and the Ghibe River Valley.</p> <p>Conclusion</p> <p>Since <it>An. arabiensis </it>shows high levels of resistance to DDT in all villages tested and varying pyrethroid resistance in Gorgora and the Ghibe River valley, precautionary measures should be taken in future vector control operations. Moreover, the status of resistance in other locations in Ethiopia and the spread of resistant gene (s) should be investigated.</p

Indoor collections of the Anopheles funestus group (Diptera: Culicidae) in sprayed houses in northern KwaZulu-Natal, South Africa

BACKGROUND: Insecticide resistance in malaria vector mosquitoes presents a serious problem for those involved in control of this disease. South Africa experienced a severe malaria epidemic during 1999/2000 due to pyrethroid resistance in the major vector Anopheles funestus. Subsequent monitoring and surveillance of mosquito populations were conducted as part of the malaria vector control programme. METHODS: A sample of 269 Anopheles funestus s.l. was collected in Mamfene, northern KwaZulu-Natal, using exit window traps in pyrethroid sprayed houses between May and June 2005. Mosquitoes were identified to species level, assayed for insecticide susceptibility, analysed for Plasmodium falciparum infectivity and blood meal source. RESULTS: Of the 220 mosquitoes identified using the rDNA PCR method, two (0.9%) were An. funestus s.s. and 218 (99.1%) Anopheles parensis. Standard WHO insecticide susceptibility tests were performed on F1 progeny from wild caught An. parensis females and a significant survival 24 h post exposure was detected in 40% of families exposed to 0.05% deltamethrin. Biochemical analysis of F1 An. parensis showed no elevation in levels/activity of the detoxifying enzyme systems when compared with an insecticide susceptible An. funestus laboratory strain. Among the 149 female An. parensis tested for P. falciparum circumsporozoite infections, 13.4% were positive. All ELISA positive specimens (n = 20) were re-examined for P. falciparum infections using a PCR assay and none were found to be positive. Direct ELISA analysis of 169 blood meal positive specimens showed > 75% of blood meals were taken from animals. All blood fed, false positive mosquito samples for the detection of sporozoites of P. falciparum were zoophilic. CONCLUSION: The combination of pyrethroid resistance and P. falciparum false-positivity in An. parensis poses a problem for vector control. If accurate species identification had not been carried out, scarce resources would have been wasted in the unnecessary changing of control strategies to combat a non-vector species

Use of alternative bioassays to explore the impact of pyrethroid resistance on LLIN efficacy

Background: There is substantial concern that the spread of insecticide resistance will render long-lasting insecticide-treated nets (LLINs) ineffective. However, there is limited evidence supporting a clear association between insecticide resistance and malaria incidence or prevalence in the field. We suggest that one reason for this disconnect is that the standard WHO assays used in surveillance to classify mosquito populations as resistant are not designed to determine how resistance might impact LLIN efficacy. The standard assays expose young, unfed female mosquitoes to a diagnostic insecticide dose in a single, forced exposure, whereas in the field, mosquitoes vary in their age, blood-feeding status, and the frequency or intensity of LLIN exposure. These more realistic conditions could ultimately impact the capacity of "resistant" mosquitoes to transmit malaria. Methods: Here, we test this hypothesis using two different assays that allow female mosquitoes to contact a LLIN as they host-seek and blood-feed. We quantified mortality after both single and multiple exposures, using seven different strains of Anopheles ranging in pyrethroid resistance intensity. Results: We found that strains classified as 1×-resistant to the pyrethroid insecticide deltamethrin in the standard WHO assay exhibited > 90% mortality over 24 h following more realistic LLIN contact. Mosquitoes that were able to blood-feed had increased survival compared to their unfed counterparts, but none of the 1×-resistant strains survived for 12 days post-exposure (the typical period for malaria parasite development within the mosquito). Mosquitoes that were 5×- and 10×-resistant (i.e. moderate or high intensity resistance based on the WHO assays) survived a single LLIN exposure well. However, only about 2-3% of these mosquitoes survived multiple exposures over the course of 12 days and successfully blood-fed during the last exposure. Conclusions: These results suggest that the standard assays provide limited insight into how resistance might impact LLIN efficacy. In our laboratory setting, there appears little functional consequence of 1×-resistance and even mosquitoes with moderate (5×) or high (10×) intensity resistance can suffer substantial reduction in transmission potential. Monitoring efforts should focus on better characterizing intensity of resistance to inform resistance management strategies and prioritize deployment of next generation vector control products.[Figure not available: see fulltext.

The infectivity of the entomopathogenic fungus Beauveria bassiana to insecticide-resistant and susceptible Anopheles arabiensis mosquitoes at two different temperatures

BACKGROUND: Control of the major African malaria vector species continues to rely extensively on the application of residual insecticides through indoor house spraying or bed net impregnation. Insecticide resistance is undermining the sustainability of these control strategies. Alternatives to the currently available conventional chemical insecticides are, therefore, urgently needed. Use of fungal pathogens as biopesticides is one such possibility. However, one of the challenges to the approach is the potential influence of varied environmental conditions and target species that could affect the efficacy of a biological 'active ingredient'. An initial investigation into this was carried out to assess the susceptibility of insecticide-susceptible and resistant laboratory strains and wild-collected Anopheles arabiensis mosquitoes to infection with the fungus Beauveria bassiana under two different laboratory temperature regimes. METHODS: Insecticide susceptibility to all four classes of insecticides recommended by WHO for vector control was tested on laboratory and wild-caught An. arabiensis, using standard WHO bioassay protocols. Mosquito susceptibility to fungus infection was tested using dry spores of B. bassiana under two temperature regimes (21 +/- 1 degrees C or 25 +/- 2 degrees C) representative of indoor conditions observed in western Kenya. Cox regression analysis was used to assess the effect of fungal infection on mosquito survival and the effect of insecticide resistance status and temperature on mortality rates following fungus infection. RESULTS: Survival data showed no relationship between insecticide susceptibility and susceptibility to B. bassiana. All tested colonies showed complete susceptibility to fungal infection despite some showing high resistance levels to chemical insecticides. There was, however, a difference in fungus-induced mortality rates between temperature treatments with virulence significantly higher at 25 degrees C than 21 degrees C. Even so, because malaria parasite development is also known to slow as temperatures fall, expected reductions in malaria transmission potential due to fungal infection under the cooler conditions would still be high. CONCLUSIONS: These results provide evidence that the entomopathogenic fungus B. bassiana has potential for use as an alternative vector control tool against insecticide-resistant mosquitoes under conditions typical of indoor resting environments. Nonetheless, the observed variation in effective virulence reveals the need for further study to optimize selection of isolates, dose and use strategy in different eco-epidemiological setting