The impact of temperature on insecticide toxicity against the malaria vectors Anopheles arabiensis and Anopheles funestus

BACKGROUND: It is anticipated that malaria elimination efforts in Africa will be hampered by increasing resistance to the limited arsenal of insecticides approved for use in public health. However, insecticide susceptibility status of vector populations evaluated under standard insectary test conditions can give a false picture of the threat, as the thermal environment in which the insect and insecticide interact plays a significant role in insecticide toxicity. METHODS: The effect of temperature on the expression of the standard WHO insecticide resistance phenotype was examined using Anopheles arabiensis and Anopheles funestus strains: a susceptible strain and the derived resistant strain, selected in the laboratory for resistance to DDT or pyrethroids. The susceptibility of mosquitoes to the pyrethroid deltamethrin or the carbamate bendiocarb was assessed at 18, 25 or 30 degrees C. The ability of the pyrethroid synergist piperonyl-butoxide (PBO) to restore pyrethroid susceptibility was also assessed at these temperatures. RESULTS: Temperature impacted the toxicity of deltamethrin and bendiocarb. Although the resistant An. funestus strain was uniformly resistant to deltamethrin across temperatures, increasing temperature increased the resistance of the susceptible An. arabiensis strain. Against susceptible An. funestus and resistant An. arabiensis females, deltamethrin exposure at temperatures both lower and higher than standard insectary conditions increased mortality. PBO exposure completely restored deltamethrin susceptibility at all temperatures. Bendiocarb displayed a consistently positive temperature coefficient against both susceptible and resistant An. funestus strains, with survival increasing as temperature increased. CONCLUSIONS: Environmental temperature has a marked effect on the efficacy of insecticides used in public health against important African malaria vectors. Caution must be exercised when drawing conclusions about a chemical's efficacy from laboratory assays performed at only one temperature, as phenotypic resistance can vary significantly even over a temperature range that could be experienced by mosquitoes in the field during a single day. Similarly, it might be inappropriate to assume equal efficacy of a control tool over a geographic area where local conditions vary drastically. Additional studies into the effects of temperature on the efficacy of insecticide-based interventions under field conditions are warranted

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.

Malaria risk and receptivity: Continuing development of insecticide resistance in the major malaria vector Anopheles arabiensis in northern KwaZulu-Natal, South Africa

Malaria incidence in South Africa is highest in the three endemic provinces: KwaZulu-Natal, Mpumalanga and Limpopo. The contribution to malaria transmission by several mosquito species, variation in their resting behaviours and low levels of insecticide resistance makes it necessary to periodically monitor Anopheles species assemblages and resistance phenotypes in vector populations. The aim of this study was therefore to assess Anopheles species assemblage in northern KwaZulu-Natal and to collect insecticide susceptibility data for An. arabiensis, the primary vector of malaria in that province. Anopheles specimens were collected from Mamfene, Jozini, northern KwaZulu-Natal from November 2019 to April 2021. Progeny of wild-collected An. arabiensis females were used for standard insecticide susceptibility tests and synergist bioassays. Anopheles arabiensis contributed 85.6% (n=11 062) of the total catches. Samples for subsequent insecticide susceptibility bioassays were selected from 212 An. arabiensis families. These showed low-level resistance to DDT, permethrin, deltamethrin, and bendiocarb, as well as full susceptibility to pirimiphos-methyl. Synergist bioassays using piperonyl butoxide and triphenyl phosphate suggest oxygenase-based pyrethroid and esterase-mediated sequestration of bendiocarb. These low levels of resistance are unlikely to be operationally significant at present. It is concluded that northern KwaZulu-Natal Province remains receptive to malaria transmission despite ongoing control and elimination interventions. This is due to the perennial presence of the major vector An. arabiensis and other secondary vector species. The continued detection of low-frequency insecticide resistance phenotypes in An. arabiensis is cause for concern and requires periodic monitoring for changes in resistance frequency and intensity.Significance:• Insecticide resistance in the major malaria vector Anopheles arabiensis in northern KwaZulu-Natal Province is cause for concern in terms of resistance management and ongoing vector control leading toward malaria elimination.• Despite ongoing control interventions, northern KwaZulu-Natal remains receptive to malaria owing to the perennial presence of several Anopheles vector species

The Role of Oxidative Stress in the Longevity and Insecticide Resistance Phenotype of the Major Malaria Vectors Anopheles arabiensis and Anopheles funestus.

Oxidative stress plays numerous biological roles, both functional and pathological. The role of oxidative stress in various epidemiologically relevant biological traits in Anopheles mosquitoes is not well established. In this study, the effects of oxidative stress on the longevity and insecticide resistance phenotype in the major malaria vector species An. arabiensis and An. funestus were examined. Responses to dietary copper sulphate and hydrogen peroxide were used as proxies for the oxidative stress phenotype by determining the effect of copper on longevity and hydrogen peroxide lethal dose. Glutathione peroxidase and catalase activities were determined colorimetrically. Oxidative burden was quantified as protein carbonyl content. Changes in insecticide resistance phenotype were monitored by WHO bioassay. Insecticide resistant individuals showed an increased capacity for coping with oxidative stress, mediated by increased glutathione peroxidase and catalase activity. This effect was observed in both species, as well as in laboratory strains and F1 individuals derived from wild-caught An. funestus mothers. Phenotypic capacity for coping with oxidative stress was greatest in strains with elevated Cytochrome P450 activity. Synergism of oxidative stress defence enzymes by dietary supplementation with haematin, 3-Amino-1, 2, 4-triazole and Sodium diethyldithiocarbamate significantly increased pyrethroid-induced mortality in An. arabiensis and An. funestus. It is therefore concluded that defence against oxidative stress underlies the augmentation of the insecticide resistance phenotype associated with multiple blood-feeding. This is because multiple blood-feeding ultimately leads to a reduction of oxidative stress in insecticide resistant females, and also reduces the oxidative burden induced by DDT and pyrethroids, by inducing increased glutathione peroxidase activity. This study highlights the importance of oxidative stress in the longevity and insecticide resistance phenotype in malaria vectors

The effect of metal pollution on the life history and insecticide resistance phenotype of the major malaria vector Anopheles arabiensis (Diptera: Culicidae).

Metal exposure is one of the commonest anthropogenic pollutants mosquito larvae are exposed to, both in agricultural and urban settings. As members of the Anopheles gambiae complex, which contains several major malaria vector species including An. arabiensis, are increasingly adapting to polluted environments, this study examined the effects of larval metal exposure on various life history traits of epidemiological importance. Two laboratory strains of An. arabiensis, SENN (insecticide susceptible) and SENN DDT (insecticide resistant), were reared in maximum acceptable toxicity concentrations, (MATC-the highest legally accepted concentration) of cadmium chloride, lead nitrate and copper nitrate. Following these exposures, time to pupation, adult size and longevity were determined. Larvae reared in double the MATC were assessed for changes in malathion and deltamethrin tolerance, measured by lethal time bottle bioassay, as well as changes in detoxification enzyme activity. As defence against oxidative stress has previously been demonstrated to affect the expression of insecticide resistance, catalase, glutathione peroxidase and superoxide dismutase activity was assessed. The relative metal toxicity to metal naïve larvae was also assessed. SENN DDT larvae were more tolerant of metal pollution than SENN larvae. Pupation in SENN larvae was significantly reduced by metal exposure, while adult longevity was not affected. SENN DDT showed decreased adult size after larval metal exposure. Adult insecticide tolerance was increased after larval metal exposure, and this effect appeared to be mediated by increased β-esterase, cytochrome P450 and superoxide dismutase activity. These data suggest an enzyme-mediated positive link between tolerance to metal pollutants and insecticide resistance in adult mosquitoes. Furthermore, exposure of larvae to metal pollutants may have operational consequences under an insecticide-based vector control scenario by increasing the expression of insecticide resistance in adults