Insecticide resistance in Culex quinquefasciatus from Zanzibar: implications for vector control programmes

Background Zanzibar has a long history of lymphatic filariasis (LF) caused by the filarial parasite Wuchereria bancrofti, and transmitted by the mosquito Culex quinquefasciatus Say. The LF Programme in Zanzibar has successfully implemented mass drug administration (MDA) to interrupt transmission, and is now in the elimination phase. Monitoring infections in mosquitoes, and assessing the potential role of interventions such as vector control, is important in case the disease re-emerges as a public health problem. Here, we examine Culex mosquito species from the two main islands to detect W. bancrofti infection and to determine levels of susceptibility to the insecticides used for vector control. Methods Culex mosquitoes collected during routine catches in Vitongoji, Pemba Island, and Makadara, Unguja Island were tested for W. bancrofti infection using PCR. Insecticide bioassays on Culex mosquitoes were performed to determine susceptibility to permethrin, deltamethrin, lambda-cyhalothrin, DDT and bendiocarb. Additional synergism assays with piperonyl butoxide (PBO) were used for lambda-cyhalothrin. Pyrosequencing was used to determine the kdr genotype and sequencing of the mitochondrial cytochrome oxidase I (mtCOI) subunit performed to identify ambiguous Culex species. Results None of the wild-caught Culex mosquitoes analysed were found to be positive for W. bancrofti. High frequencies of resistance to all insecticides were found in Wete, Pemba Island, whereas Culex from the nearby site of Tibirinzi (Pemba) and in Kilimani, Unguja Island remained relatively susceptible. Species identification confirmed that mosquitoes from Wete were Culex quinquefasciatus. The majority of the Culex collected from Tibirinzi and all from Kilimani could not be identified to species by molecular assays. Two alternative kdr alleles, both resulting in a L1014F substitution were detected in Cx. quinquefasciatus from Wete with no homozygote susceptible detected. Metabolic resistance to pyrethroids was also implicated by PBO synergism assays. Conclusions Results from the xenomonitoring are encouraging for the LF programme in Zanzibar. However, the high levels of pyrethroid resistance found in the principle LF vector in Pemba Island will need to be taken into consideration if vector control is to be implemented as part of the elimination programme

The Dynamics of Pyrethroid Nesistance in Anopheles Arabiensis from Zanzibar and an Assessment of the Underlying Genetic Basis.

The emergence of pyrethroid resistance in the malaria vector, Anopheles arabiensis, threatens to undermine the considerable gains made towards eliminating malaria on Zanzibar. Previously, resistance was restricted to the island of Pemba while mosquitoes from Unguja, the larger of the two islands of Zanzibar, were susceptible. Here, we characterised the mechanism(s) responsible for resistance on Zanzibar using a combination of gene expression and target-site mutation assays. WHO resistance bioassays were conducted using 1-5d old adult Anopheles gambiae s.l. collected between 2011 and 2013 across the archipelago. Synergist assays with the P450 inhibitor piperonyl-butoxide were performed in 2013. Members of the An. gambiae complex were PCR-identified and screened for target-site mutations (kdr and Ace-1). Gene expression in pyrethroid resistant An. arabiensis from Pemba was analysed using whole-genome microarrays. Pyrethroid resistance is now present across the entire Zanzibar archipelago. Survival to the pyrethroid lambda-cyhalothrin in bioassays conducted in 2013 was 23.5-54.3% on Unguja and 32.9-81.7% on Pemba. We present evidence that resistance is mediated, in part at least, by elevated P450 monoxygenases. Whole-genome microarray scans showed that the most enriched gene terms in resistant An. arabiensis from Pemba were associated with P450 activity and synergist assays with PBO completely restored susceptibility to pyrethroids in both islands. CYP4G16 was the most consistently over-expressed gene in resistant mosquitoes compared with two susceptible strains from Unguja and Dar es Salaam. Expression of this P450 is enriched in the abdomen and it is thought to play a role in hydrocarbon synthesis. Microarray and qPCR detected several additional genes putatively involved in this pathway enriched in the Pemba pyrethroid resistant population and we hypothesise that resistance may be, in part, related to alterations in the structure of the mosquito cuticle. None of the kdr target-site mutations, associated with pyrethroid/DDT resistance in An. gambiae elsewhere in Africa, were found on the islands. The consequences of this resistance phenotype are discussed in relation to future vector control strategies on Zanzibar to support the ongoing malaria elimination efforts on the islands

Efficacy, persistence and vector susceptibility to pirimiphos-methyl (Actellic® 300CS) insecticide for indoor residual spraying in Zanzibar

Background Indoor residual spraying (IRS) of households with insecticide is a principal malaria vector control intervention in Zanzibar. In 2006, IRS using the pyrethroid lambda-cyhalothrine was introduced in Zanzibar. Following detection of pyrethroid resistance in 2010, an insecticide resistance management plan was proposed, and IRS using bendiocarb was started in 2011. In 2014, bendiocarb was replaced by pirimiphos methyl. This study investigated the residual efficacy of pirimiphos methyl (Actellic® 300CS) sprayed on common surfaces of human dwellings in Zanzibar. Methods The residual activity of Actellic 300CS was determined over 9 months through bioassay tests that measured the mortality of female Anopheles mosquitoes, exposed to sprayed surfaces under a WHO cone. The wall surfaces included; mud wall, oil or water painted walls, lime washed wall, un-plastered cement block wall and stone blocks. Insecticide susceptibility testing was done to investigate the resistance status of local malaria vectors against Actellic 300CS using WHO protocols; Anopheline species were identified using PCR methods. Results Baseline tests conducted one-day post-IRS revealed 100 % mortality on all sprayed surfaces. The residual efficacy of Actellic 300CS was maintained on all sprayed surfaces up to 8 months post-IRS. However, the bioassay test conducted 9 months post-IRS showed the 24 h mortality rate to be ≤80 % for lime wash, mud wall, water paint and stone block surfaces. Only oil paint surface retained the recommended residual efficacy beyond 9 months post-IRS, with mortality maintained at ≥97 %. Results of susceptibility tests showed that malaria vectors in Zanzibar were fully (100 %) susceptible to Actellic 300CS. The predominant mosquito vector species was An. arabiensis (76.0 %) in Pemba and An. gambiae (83.5 %) in Unguja. Conclusion The microencapsulated formulation of pirimiphos methyl (Actellic 300CS) is a highly effective and appropriate insecticide for IRS use in Zanzibar as it showed a relatively prolonged residual activity compared to other products used for the same purpose. The insecticide extends the residual effect of IRS thereby making it possible to effectively protect communities with a single annual spray round reducing overall costs. The insecticide proved to be a useful alternative in insecticide resistance management plans

Islands and Stepping-Stones: Comparative Population Structure of Anopheles gambiae sensu stricto and Anopheles arabiensis in Tanzania and Implications for the Spread of Insecticide Resistance

Population genetic structures of the two major malaria vectors Anopheles gambiae s.s. and An. arabiensis, differ markedly across Sub-Saharan Africa, which could reflect differences in historical demographies or in contemporary gene flow. Elucidation of the degree and cause of population structure is important for predicting the spread of genetic traits such as insecticide resistance genes or artificially engineered genes. Here the population genetics of An. gambiae s.s. and An. arabiensis in the central, eastern and island regions of Tanzania were compared. Microsatellite markers were screened in 33 collections of female An. gambiae s.l., originating from 22 geographical locations, four of which were sampled in two or three years between 2008 and 2010. An. gambiae were sampled from six sites, An. arabiensis from 14 sites, and both species from two sites, with an additional colonised insectary sample of each species. Frequencies of the knock-down resistance (kdr) alleles 1014S and 1014F were also determined. An. gambiae exhibited relatively high genetic differentiation (average pairwise FST = 0.131), significant even between nearby samples, but without clear geographical patterning. In contrast, An. arabiensis exhibited limited differentiation (average FST = 0.015), but strong isolation-by-distance (Mantel test r = 0.46, p = 0.0008). Most time-series samples of An. arabiensis were homogeneous, suggesting general temporal stability of the genetic structure. An. gambiae populations from Dar es Salaam and Bagamoyo were found to have high frequencies of kdr 1014S (around 70%), with almost 50% homozygote but was at much lower frequency on Unguja Island, with no. An. gambiae population genetic differentiation was consistent with an island model of genetic structuring with highly restricted gene flow, contrary to An. arabiensis which was consistent with a stepping-stone model of extensive, but geographically-restricted gene flow

Neighbour-joining tree based on linearised F_ST.

<p>Sample names with the prefix GA are <i>An. gambiae s.s.</i>, and those with the prefix AR are <i>An. arabiensis</i>. Samples labelled IFAKARA are from insectary colonies. Within each species, samples identified as distinct clusters by BAPS are circled; others fall within a single cluster in each species.</p

F_IS and Lositan values for <i>An. gambiae s.s</i>.(A) and <i>An. arabiensis</i> (B).

<p>FIS values in bold are significantly greater or larger than expected (following Bonferroni correction).</p><p>Lositan P values in red indicate loci showing patterns of differentiation exceeding neutral expectations; these loci were excluded.</p><p>F_IS and Lositan values for <i>An. gambiae s.s</i>.(A) and <i>An. arabiensis</i> (B).</p

Individual-based BAPS spatially-conditioned clustering.

<p>Cluster identification numbers (k<i>n</i>) are shown, with number of individuals after the underscore.</p

Genetic diversity in (A) <i>An. gambiae s.s.</i> and (B) <i>An. arabiensis</i>.

<p>Each plot shows the mean expected heterozygosity or allelic richness across loci with 95% confidence intervals.</p

<i>Kdr</i> L1014S in <i>An. gambiae s.s.</i>

<p>Serine allele (filled triangles) and serine homozygote (open diamonds) frequencies are plotted against a principal component reflecting location (explaining 87% of variation in latitude and longitude).</p

Map of central-eastern Tanzania showing sampling sites.

<p>Map of central-eastern Tanzania showing sampling sites.</p