Insecticide resistance in the malaria vector Anopheles arabiensis in Mamfene , KwaZulu-Natal

HOW TO CITE: Brooke BD, Robertson L, Kaiser ML, Raswiswi E, Munhenga G, Venter N, et al. Insecticide resistance in the malaria vector Anopheles arabiensis in Mamfene, KwaZulu-Natal. S Afr J Sci. 2015;111(11/12), Art. #2015-0261, 3 pages. http://dx.doi.org/10.17159/ sajs.2015/20150261 The control of malaria vector mosquitoes in South Africa’s affected provinces is primarily based on indoor spraying of long-lasting residual insecticides. The primary vectors in South Africa are Anopheles arabiensis and An. funestus. South Africa’s National Malaria Control Programme has adopted a malaria elimination agenda and has scaled up vector control activities accordingly. However, despite these plans, local transmission continues and is most likely because of outdoor feeding by populations of An. arabiensis. An outdoor Anopheles surveillance system has been set up in three sections of the Mamfene district in northern KwaZuluNatal in order to assess the extent of outdoor resting An. arabiensis in Mamfene and to assess the current insecticide susceptibility status of this population. According to WHO criteria, the An. arabiensis samples tested showed evidence of resistance to deltamethrin (pyrethroid), DDT (organochlorine) and bendiocarb (carbamate), and full susceptibility to the organophosphates pirimiphos-methyl and fenitrothion. Pre-exposure to piperonyl butoxide completely nullified the deltamethrin resistance otherwise evident in these samples, supporting previous studies implicating monooxygenase-based detoxification as the primary mechanism of pyrethroid resistance. The data presented here affirm the presence of pyrethroid and DDT resistance previously detected in this population and also indicate the comparatively recent emergence of resistance to the carbamate insecticide bendiocarb. These data show that special attention and commitment needs to be given to the principles of insecticide resistance management as well as to investigations into alternative control techniques designed to target outdoor-resting An. arabiensis in northern KwaZulu-Natal.


Introduction
The control of malaria vector mosquitoes in South Africa's affected provinces is primarily based on indoor spraying of long-lasting residual insecticides. 1The indoor residual spraying (IRS) method has been the mainstay of malaria vector control in South Africa since the late 1940s and has remained effective owing to carefully co-ordinated IRS programmes in South Africa's Limpopo, Mpumalanga and KwaZulu-Natal Provinces. 2ly Anopheles mosquitoes can transmit human malaria parasites and the primary vectors in South Africa are Anopheles arabiensis and An.funestus. 1Of these, An. funestus is almost entirely anthropophilic (human biting), endophagic (indoor feeding) and endophilic (indoor resting). 3These characteristics make this species especially susceptible to control by IRS, assuming that the insecticide employed for this purpose is effective against the target An.funestus population.Control by IRS means that the mosquitoes must retain complete or near complete susceptibility to the insecticide class being used, which can only be ascertained by regular monitoring and surveillance.The malaria epidemic experienced in South Africa during the period 1996-2000 was largely the result of the development of resistance to pyrethroid insecticides in populations of this species in northern KwaZulu-Natal and Mpumalanga which led to vector control failure. 1,2Control was re-established using a mosaic resistance management system which was later drafted into a World Health Organization (WHO) document --the Global Plan for Insecticide Resistance Management (GPIRM). 4South Africa currently subscribes to the principles outlined in GPIRM as part of its malaria elimination agenda. 5However, despite these plans and the scaling up of vector control activities in South Africa, local transmission continues, most likely because of outdoor transmission by populations of An. arabiensis.Unlike An. funestus, An. arabiensis has evolved substantial behavioural plasticity and will feed and rest indoors and outdoors, and will feed on humans as well as livestock, especially bovines. 3Anopheles arabiensis is therefore substantially less susceptible to control by IRS.
Recently, a project was launched to assess the feasibility of the sterile insect technique for malaria vector control in South Africa, with special emphasis on controlling outdoor transmission by An. arabiensis. 6As part of the baseline survey linked to this project, an outdoor Anopheles surveillance system has been set up in three sections of the Mamfene district in northern KwaZulu-Natal.This surveillance system has enabled recent assessments of insecticide resistance in outdoor-resting An. arabiensis in Mamfene as a follow-up to the discovery of pyrethroid resistance in this region in 2005. 7

Methods
In order to assess the current insecticide susceptibility status of outdoor resting An. arabiensis in Mamfene, adult Anopheles mosquitoes were collected from outdoor-placed ceramic pots 8 and modified plastic buckets deployed in 20 households in Mamfene Sections 2, 8 and 9 during March and April 2015.These collections were transported live to the Botha De Meillon insectary facility at the National Institute for Communicable Diseases (NICD) in Johannesburg.Blood-fed female specimens were individually placed in egg-laying vials so that eggs could be harvested and reared by family.All wild-caught female individuals, including those that produced eggs, were identified to species group using morphological keys 9,10 and to species by standard PCR 11 .A total of 35 families identified as An.arabiensis was pooled and the F1 progeny were reared to adults under standard insectary conditions of 25 °C and 80% relative humidity. 12ttp://www.sajs.co.za November/December 2015 Samples of 2-5-day-old female adult F1 progeny were assessed for their susceptibility to diagnostic concentrations of a range of insecticides according to the standard WHO bioassay method. 13Controls included samples of 2-4-day-old F1 male adults exposed to untreated papers.
In addition, a subset of samples was used to assess the effect of preexposure to the insecticide synergist piperonyl butoxide (PBO) on the expression of pyrethroid resistance according to a method previously described by Brooke et al. 14
The first assessments of resistance in An. arabiensis at Mamfene were conducted in 1996 and no resistance phenotypes were recorded. 15However, subsequent samples collected in 2002 indicated the emergence of resistance to DDT 16 which was again recorded in 2005 together with the first indication of pyrethroid resistance 7 .The 2015 data presented here affirm the presence of pyrethroid and DDT resistance in this population, albeit at a low frequency, and also indicate the comparatively recent emergence of resistance to the carbamate insecticide bendiocarb.The PBO exposure data support previous analyses implicating monooxygenasemediated detoxification as the primary mode of pyrethroid resistance in An. arabiensis at Mamfene, 7,17,18 because PBO enhances insecticide toxicity by providing an alternative substrate for monooxygenases.PBO pre-exposure assays can therefore be used to elucidate monooxygenasebased resistance mechanisms.

Conclusion
South Africa's 1996 to 2000 malaria epidemic illustrates the effect that a single insecticide resistance phenotype (pyrethroid resistance in An. funestus) can have on an IRS-based vector control programme. 2,19,20he occurrence of multiple vector species carrying multiple resistance mecha nisms coupled to ongoing outdoor transmission in northern KwaZulu-Natal means that special attention and commitment needs to be given to the principles of insecticide resistance management as outlined in the GPIRM document 4 as well as to investigations into alternative control techniques designed to target outdoor-resting An. arabiensis.

Table 1:
Mean percentage mortalities recorded for samples of 2-5-day-old F1 Anopheles arabiensis female adults following exposure to listed insecticides by class.Mortalities were recorded 24 h post exposure.The number of replicates, sample size (n) and standard error (s.e.) are given for each insecticide treatment.Samples by treatment are categorised as resistant (R) or susceptible (S) according to standard criteria. 13The control was exposure of 2-4-day-old F1 Anopheles arabiensis male adults to untreated papers.

Table 2 :
Mean percentage mortalities recorded for samples of 2-6-day-old F1 Anopheles arabiensis male adults (M) or female adults (F) following exposure to either deltamethrin (0.05%), piperonyl butoxide (PBO) (4%), deltamethrin + PBO or untreated control papers.Mortalities were recorded 24 h post exposure.The number of replicates, sample size (n) and standard error (s.e.) are given for each treatment.Mortalities following exposure to PBO and control papers were negligible.There is a significant difference in mean mortality between the deltamethrin and deltamethrin + PBO treatments 24 h post exposure (paired sample t-test: d.f.=1, t=15.65,p=0.04).