INAUGURAL ARTICLE by a Recently Elected Academy Member:Impact of pyrethroid resistance on operational malaria control in Malawi

The impact of insecticide resistance on insect-borne disease programs is difficult to quantify. The possibility of eliminating malaria in high-transmission settings is heavily dependent on effective vector control reducing disease transmission rates. Pyrethroids are the dominant insecticides used for malaria control, with few options for their replacement. Their failure will adversely affect our ability to control malaria. Pyrethroid resistance has been selected in Malawi over the last 3 y in the two major malaria vectors Anopheles gambiae and Anopheles funestus, with a higher frequency of resistance in the latter. The resistance in An. funestus is metabolically based and involves the up-regulation of two duplicated P450s. The same genes confer resistance in Mozambican An. funestus, although the levels of up-regulation differ. The selection of resistance over 3 y has not increased malaria transmission, as judged by annual point prevalence surveys in 1- to 4-y-old children. This is true in areas with long-lasting insecticide-treated nets (LLINs) alone or LLINs plus pyrethroid-based insecticide residual spraying (IRS). However, in districts where IRS was scaled up, it did not produce the expected decrease in malaria prevalence. As resistance increases in frequency from this low initial level, there is the potential for vector population numbers to increase with a concomitant negative impact on control efficacy. This should be monitored carefully as part of the operational activities in country.The push for malaria elimination and eventual eradication will be heavily dependent on our ability to reduce disease transmission. A recent editorial suggests that we have the tools to take on this challenge in African malaria heartlands (1). This is predicated on ensuring that vector control prevention and drug treatment tools are fully deployed, reaching every person at risk. There will need to be improved delivery of these tools and better clinical management of malaria cases. In highly endemic areas our ability to reduce malaria transmission will be dependent on vector control, before the focus can shift to killing the parasite in infected people. Two forms of vector control, indoor residual spraying (IRS) and the distribution of long-lasting insecticide-treated nets (LLINs) have been demonstrated to reduce transmission when properly deployed against insecticide susceptible mosquito populations. The use of both interventions has dramatically increased since 2000 in many malaria endemic countries, with increased donor funding to attain the Roll Back Malaria targets and support the malaria elimination agenda (2).IRS and LLINs function by reducing the female mosquito daily survival rate and human biting frequency. Pyrethroids are the only insecticides recommended for use on LLINs, and only four chemical classes of insecticides that attack two target sites are available for IRS, and again pyrethroids dominate the IRS market. Resistance to pyrethroids has been selected in Anopheles gambiae and Anopheles funestus, the major African malaria vectors, although the frequency and level (fold) resistance conferred can vary dramatically. The impact of this resistance on the ability of either control intervention to reduce disease transmission is poorly understood, and current monitoring and evaluation practices are not sufficiently robust to assess this unless catastrophic failures occur. The perceived threat of pyrethroid resistance is now sufficiently high for the World Health Organization (WHO) to convene an international multidonor effort to counteract this.Operationally significant pyrethroid resistance has the potential to limit effective malaria control, owing to the small number of alternative public health insecticides. Pyrethroid resistance in malaria vectors has increased dramatically over the last decade (3, 4), particularly in Africa, where the bulk of malaria-related mortality occurs. Typically resistance is monitored by bioassays, for which the WHO has defined a diagnostic dosage for each insecticide that kills susceptible anopheline mosquitoes (5). Mosquitoes surviving the diagnostic dosage are an indication that resistance has been selected and that an operational problem may be developing, but bioassays alone do not signify control failure.Little operational monitoring of the underlying mechanisms of resistance occurs. Two mechanisms are predominantly responsible for insecticide resistance: changes in the insecticide target site, reducing binding of the insecticide, and increases in the rate at which the insecticide is metabolized (6). Information on the resistance mechanisms is more predictive than bioassays, providing information on the level of resistance and potential cross-resistance between insecticides. For example, two common mutations in the sodium channel convey low-level resistance to pyrethroids and higher-level resistance to dichlorodiphenyltrichloroethane (DDT) in An. gambiae (7, 8), whereas a cytochrome P450-based metabolic regulatory mechanism conveys very high-level pyrethroid and low-level carbamate resistance in An. funestus (9).Vector control interventions are being rapidly scaled up in Malawi, where malaria is highly endemic. Malaria accounts for 34% of all outpatient hospital visits and is the main cause of hospital admissions in children aged <5 y (10). Before 2007 sporadic WHO bioassays were undertaken, which indicated that the two major malaria vectors, An. gambiae and An. funestus, remained fully susceptible to pyrethroids. In 2007 pyrethroid-impregnated LLINs were distributed through antenatal and under-5 clinics at district and central hospitals countrywide. The numbers distributed were sufficient to achieve the Roll Back Malaria targets of 80% of pregnant women and children aged <5 y sleeping under a treated net. In 2008, a pilot study of IRS with the pyrethroid lambda cyhalothrin (ICON, Syngenta) was initiated in Nkhota Khota District, supported by the President’s Malaria Initiative (PMI). The initial program targeted 26,950 houses, and was expanded to 74,772 houses in 2009. Approximately 4 million LLINs were procured and ∼2 million distributed during this time. In 2010 the PMI-supported IRS was expanded to cover the whole of Nkohta Khota district, and the Malawian Ministry of Health supported IRS in a further six districts.A series of sentinel sites were established during this period to track the effect of this rapid increase in insecticide selection pressure on the local vectors and assess any impact on malaria transmission. This was particularly pertinent owing to the high levels of pyrethroid resistance reported in the southern part of neighboring Mozambique in An. funestus, which had prompted a switch from pyrethroids to carbamates or DDT for IRS in the Lubombo Spatial Development Initiative area of Mozambique (11, 12).     

Spatio-Temporal Patterns in kdr Frequency in Permethrin and DDT Resistant Anopheles gambiae s.s. from Uganda

The planned upscaling of vector control strategies requires insight into the epidemiological consequences of vector resistance. Therefore, the pyrethroid and DDT resistance status of Anopheles gambiae s.l. was assessed in Uganda from 2004 to 2006, and spatial and seasonal variations in knockdown resistance (kdr) frequencies were analyzed in terms of epidemiological significance. Anopheles gambiae s.l. was DDT and pyrethroid resistant in central and eastern Uganda. The L1014S kdr allele frequencies varied from 3% to 48% in An. gambiae s.s. Although the homozygous resistant genotype was the most prevalent genotype among survivors, the genotypes could not entirely explain the bioassay results. In the dry season, the kdr frequency was significantly higher in Plasmodium falciparum-infected mosquitoes, indicating that mosquitoes bearing a kdr mutation have a better adult survival, hence a higher likelihood of becoming infectious. This study showed that kdr might have an epidemiological impact that could jeopardize the vector control strategies.     

Diurnal biting of malaria mosquitoes in the Central African Republic indicates residual transmission may be “out of control”

Malaria control interventions target nocturnal feeding of the Anopheles vectors indoors to reduce parasite transmission. Mass deployment of insecticidal bed nets and indoor residual spraying with insecticides, however, may induce mosquitoes to blood-feed at places and at times when humans are not protected. These changes can set a ceiling to the efficacy of these control interventions, resulting in residual malaria transmission. Despite its relevance for disease transmission, the daily rhythmicity of Anopheles biting behavior is poorly documented, most investigations focusing on crepuscular hours and nighttime. By performing mosquito collections 48-h around the clock, both indoors and outdoors, and by modeling biting events using circular statistics, we evaluated the full daily rhythmicity of biting in urban Bangui, Central African Republic. While the bulk of biting by Anopheles gambiae, Anopheles coluzzii, Anopheles funestus, and Anopheles pharoensis occurred from sunset to sunrise outdoors, unexpectedly ∼20 to 30% of indoor biting occurred during daytime. As biting events did not fully conform to any family of circular distributions, we fitted mixtures of von Mises distributions and found that observations were consistent with three compartments, corresponding indoors to populations of early-night, late-night, and daytime-biting events. It is not known whether these populations of biting events correspond to spatiotemporal heterogeneities or also to distinct mosquito genotypes/phenotypes belonging consistently to each compartment. Prevalence of Plasmodium falciparum in nighttime- and daytime-biting mosquitoes was the same. As >50% of biting occurs in Bangui when people are unprotected, malaria control interventions outside the domiciliary environment should be envisaged.

Transmission of parasites of the genus Plasmodium that are the causative agents of human malaria is considered to occur mainly from sunset to sunrise, when their vectors, mosquitoes of the genus Anopheles, feed on human hosts that are at rest or asleep (1, 2). This principle is so firmly established that sampling protocols to measure the strength of transmission usually disregard diurnal Anopheles feeding (3). This has not always been so: sampling routines implemented in the early 20th century by medical entomologists generally covered the whole 24-h biting cycle of different mosquito species (4). It is generally assumed that nighttime blood-feeding evolved because hosts are less active at this time, so that mosquitoes incur a lower risk of being swatted or chased away, enabling higher feeding success (2). Human hosts, however, most often rest indoors; many human-biting anophelines, therefore, penetrate inside households in order to have access to and feed on humans. Current mainstay vector control tactics exploit these behaviors in order to reduce transmission by means of two interventions: protecting humans under long-lasting insecticidal bed nets (LLINs), and spraying houses with residual insecticides (i.e., indoor residual spraying, IRS) (5). Additional benefits of IRS come from the habit of some malaria vectors to rest inside dwellings, using them as refugia either before or after blood-feeding (1).Despite the incontestable success of these control interventions in reducing the burden of malaria (6), a plateau in the incidence rate of malaria cases has been observed in Africa during the last years (7). This could be at least in part explained by the intensive selection pressure put up by insecticides (8). Indeed, mutations conferring insecticide resistance have rapidly emerged (9), and resistance to different classes of insecticides is presently widespread in most malaria vector populations (10, 11). Moreover, there is evidence that some vector populations are changing their behavior in response to control interventions by feeding progressively more at places and at times when humans are less likely to be protected (12, 13). So far, behavioral modifications have resulted in more biting during the evening and early morning and out-of-doors (14–21). Recent studies reporting significant amounts of daytime biting, however, remain somewhat anecdotal, with few exceptions. Overall, these modifications increase the window of opportunity for human–vector contact, producing what is called residual malaria transmission (22, 23): this expression implies that transmission will persist even if LLINs and IRS are fully effective. It is feared, therefore, that the malaria elimination target set upon current control interventions may be compromised in the long term by residual malaria transmission (24). Countering this phenomenon should be based on better knowledge of the biology and behavior of the vectors, with the aim to develop more suitable interventions (12, 23, 25).In the Central African Republic, malaria remains a major public health problem and the main cause of deaths among children <5 y old (26). In 2006, the National Malaria Control Program implemented the first phase of the Global Fund Program for Malaria based on free distribution of LLINs to pregnant women and children <5 y old, with moderate results (26, 27). A new campaign of mass LLIN distribution was deployed in 2015. Yet, malaria incidence was not significantly reduced (28). Several studies reported the presence in the country of populations of the major malaria vectors Anopheles gambiae, Anopheles coluzzii, and Anopheles funestus that are resistant to pyrethroids, the class of insecticides used for impregnation of bed nets (29–31). It is not known, however, the degree to which insecticide resistance is responsible for such moderate reductions in malaria incidence in this country, above and beyond what can be accounted for by a national health system weakened by years of civil war and unrest.In order to appreciate the potential of residual malaria transmission in this epidemiological context, we investigated the biting behavior of the malaria vectors occurring in urban settings of Bangui, Central African Republic. We implemented two significant modifications to the sampling protocol and analytical procedures that are usually applied in this kind of investigation. First, the sampling plan consisted of monthly sessions of 48-h around-the-clock collections of mosquitoes coming to feed on human hosts both indoors and outdoors. Second, the daily rhythmicity of the observed biting events was analyzed quantitatively using a circular statistics framework that models these events on a circumference rather than along the usual linear representation (32–34). Unexpectedly, we found that 20 to 30% of malaria vector biting occurred at full daytime indoors. These results suggest that current vector control interventions may not be enough to achieve sufficient reductions in malaria transmission in Bangui. Perhaps even more significant, these observations suggest that Anopheles mosquitoes may have the potential to achieve fundamental modifications in the temporal organization and circadian control of their feeding behavior, with major impacts on malaria control strategies in Africa. We elaborate these results as follows.     

DDT-based indoor residual spraying suboptimal for visceral leishmaniasis elimination in India

Indoor residual spraying (IRS) is used to control visceral leishmaniasis (VL) in India, but it is poorly quality assured. Quality assurance was performed in eight VL endemic districts in Bihar State, India, in 2014. Residual dichlorodiphenyltrichloroethane (DDT) was sampled from walls using Bostik tape discs, and DDT concentrations [grams of active ingredient per square meter (g ai/m²)] were determined using HPLC. Pre-IRS surveys were performed in three districts, and post-IRS surveys were performed in eight districts. A 20% threshold above and below the target spray of 1.0 g ai/m² was defined as “in range.” The entomological assessments were made in four districts in IRS and non-IRS villages. Vector densities were measured: pre-IRS and 1 and 3 mo post-IRS. Insecticide susceptibility to 4% DDT and 0.05% deltamethrin WHO-impregnated papers was determined with wild-caught sand flies. The majority (329 of 360, 91.3%) of pre-IRS samples had residual DDT concentrations of <0.1 g ai/m². The mean residual concentration of DDT post-IRS was 0.37 g ai/m²; 84.9% of walls were undersprayed, 7.4% were sprayed in range, and 7.6% were oversprayed. The abundance of sand flies in IRS and non-IRS villages was significantly different at 1 mo post-IRS only. Sand flies were highly resistant to DDT but susceptible to deltamethrin. The Stockholm Convention, ratified by India in 2006, calls for the complete phasing out of DDT as soon as practical, with limited use in the interim where no viable IRS alternatives exist. Given the poor quality of the DDT-based IRS, ready availability of pyrethroids, and susceptibility profile of Indian sand flies, the continued use of DDT in this IRS program is questionable.Leishmaniases are diseases caused by protozoan parasites that are transmitted to humans through the bites of infected female sand flies. The visceral form of leishmaniasis (VL) is endemic on the Indian subcontinent and in East Africa and Brazil, and 90% of the estimated 200–400,000 annual cases come from people living in these countries (1).An estimated 200 million people are at risk for VL, of whom 65 million live in India (2). The majority of Indian VL cases occur in the northeastern states, predominantly in Bihar (3, 4). The numbers of VL cases reported in India are based on passive case reporting, and therefore may be an underestimation (2, 5); however, regardless, advances in VL diagnostic tests and improved treatment modalities have ensured that the number of deaths due to VL in India has gradually decreased over the past 5 y (6).VL in India is caused by Leishmania donovani and is only transmitted by the female sand fly, Phlebotomus argentipes; no animal reservoir exists (2, 7). During the period of 1953–1962, indoor residual spraying (IRS) undertaken by the Indian national malaria program using dichlorodiphenyltrichloroethane (DDT) at 1 grams of active ingredient per square meter (g ai/m²) for malaria control had the beneficial side effect of significantly reducing the number of VL cases in India. A similar effect on VL due to IRS for malaria control was also seen in Bangladesh from 1961–1970 (8). In India, VL did not reappear in areas such as Assam until the late 1970s (9). This inadvertent control of VL led to the adoption of IRS by the Indian VL elimination program as the main focus for P. argentipes control, and two periods of IRS using DDT at 1 g ai/m² during 1977–1979 and 1992–1995 both saw sharp decreases in the number of VL cases reported (6).In India, IRS is performed by spraying houses and animal shelters to control the endophilic and exophagic vector P. argentipes (10). Although the effect of IRS on P. argentipes is based on limited data (11–13), a cluster randomized trial performed in India, Bangladesh, and Nepal demonstrated that IRS using DDT reduced the indoor abundance of P. argentipes by 72.4% in intervention clusters compared with controls; this effect was greater than the effect of environmental modification or the use of long-lasting insecticide-treated nets (42.0% and 43.7%, respectively) (14). Older models also predict that IRS is capable of achieving VL elimination, provided that sand fly densities are reduced by 67% (15). Given that effective prevention through vector control and rapid diagnosis and treatment methodologies exist (7, 16), elimination of VL in this region should therefore be technically feasible. However, in order for the predicted outcomes of IRS to become a reality, the IRS itself must be of sufficient quality to achieve an impact (17, 18).Strong regional political support for VL elimination was established through a tripartite agreement between Bangladesh, India, and Nepal that was signed in 2005 with the aim of reducing the annual incidence of VL and post–kala-azar dermal leishmaniasis (PKDL) to less than one case per 10,000 population at the subdistrict level by 2015 (19). This program was a four-phase process to interrupt transmission, enhance early diagnosis and treatment, and improve health education (20). The tripartite agreement also aimed to standardize IRS in the region, as recommended by the regional technical advisory group (RTAG) (21) (

Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions

Pyrethroid insecticides are used to control diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity-based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid-metabolizing and nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b₅ knockout mouse livers to validate P450 activation and establish the role for b₅ in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or “pyrethrome.” Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450–insecticide interactions and aiding the development of unique tools for disease control.Pyrethroids are synthetic analogs of pyrethrins, botanical chemicals derived from chrysanthemum flowers (1). They are highly potent insecticides with low mammalian toxicity that are used worldwide in ∼3,500 registered products in agricultural, medicinal, veterinary, and public health sectors. Importantly, they are the only class of insecticide recommended for insecticide-treated nets for malaria control. More than 254 million insecticide-treated nets were distributed across Africa between 2008–2010 (2). Similar to antibiotics, pyrethroids are critical for controlling a diverse spectrum of diseases. Unfortunately, similar to antibiotics, such intense exposure affects health and drives the rapid evolution of insecticide resistance (3).Pyrethroids are structurally highly diverse (4) but share a common architecture comprising a cyclopropane acid group coupled to an alcohol moiety, as exemplified by deltamethrin (Fig. 1A). Traditionally, they are divided into two classes (type 1 and type 2), depending on the absence (type 1) or presence (type 2) of an α-cyano group (Fig. 1B). Pyrethroids work by blocking the voltage-gated sodium channels, causing paralysis in arthropods, and resulting in death (3). Resistance is commonly associated with target site modification or metabolic resistance, in which increased rates of biotransformation, generally by P450s, esterases, and GSTs, reduce toxic potency (3).Open in a separate windowFig. 1.Conversion of deltamethrin into PyABPs. (A) Structure of deltamethrin with constituent acid and alcohol moieties. Primary sites of P450 hydroxylation are indicated by bold arrows at the 2′ and 4′ positions, and minor routes of hydroxylation are indicated with open arrows (1). (B) Conversion of deltamethrin to a PyABPP involves the addition of an alkyne warhead and a clickable handle. The structures of the general probe and the PyABPs synthesized are illustrated. Alkyne warhead groups were located in the 2′ or 4′ positions, whereas alkyne click handles replaced the cyano group (type 1) or terminal bromides (type 2). The general P450 probe 2-EN is boxed parallel to its type 1 pyrethroid analog, P2.Although the toxicity and metabolism of pyrethroids in mammals and insects have been extensively characterized (1), the role of specific enzymes and pathways involved in pyrethroid clearance is unclear. In insects, P450s are key enzymes involved with metabolic degradation, with constitutive overexpression of specific P450s leading to pyrethroid resistance (5, 6). Similarly, in mammals, the toxic potency of pyrethroids is inversely related to their rates of metabolic elimination (7), with both P450 oxidation and carboxyl esterase-mediated hydrolysis playing major roles. Humans have 57 P450 genes, rodents ∼80 P450 genes, and insects up to ∼200 P450 genes (8). Where genome information exists, genetic and microarray-based studies of pyrethroid-resistant versus susceptible populations have been used to identify P450s potentially capable of pyrethroid metabolism (3, 5). However, relatively few P450s have been functionally validated through recombinant P450 expression. Thus, probes able to identify pyrethroid-metabolizing enzymes directly would aid our understanding of the fundamental processes of insecticide–organism interactions, expanding our understanding of the risks of pyrethroid exposure to mammals and the enzymatic mechanisms of metabolism and resistance used by insects.Activity-based protein profiling (ABPP) has been described for the functional profiling of P450s (9, 10). The activity-based probes (ABPs) work in a mechanism-dependent manner to covalently label P450s, whereby the labeling events are detectable by adding a fluorescent reporter group via copper-catalyzed azide-alkyne cycloaddition (“click chemistry”) onto the probe−P450 adducts (9, 10). Furthermore, affinity tags can also be incorporated to pull down and identify probe–P450 adducts. The major advantage of ABPPs is their ability to directly assess enzyme activity. In this article, we have designed and synthesized a group of seven pyrethroid mimetic ABPs (PyABPs) on the basis of the deltamethrin scaffold (Fig. 1B) for the targeted identification of pyrethroid-metabolizing P450s in highly divergent organisms. We have investigated their reactivity profiles against pyrethroid metabolizing and nonmetabolizing recombinant mosquito P450s and mouse and rat liver microsomes. We show that PyABPs can be used to reveal pyrethroid structure–activity relationships, and they also have been used to identify pyrethroid-reactive P450s and related detoxification enzymes in rat liver microsomes, demonstrating their potential for directly assessing pyrethroid-metabolizing enzyme activity.     

Susceptibility status of malaria vectors to insecticides commonly used for malaria control in Tanzania

Objective The aim of the study was to monitor the insecticide susceptibility status of malaria vectors in 12 sentinel districts of Tanzania. Methods WHO standard methods were used to detect knock‐down and mortality in the wild female Anopheles mosquitoes collected in sentinel districts. The WHO diagnostic doses of 0.05% deltamethrin, 0.05% lambdacyhalothrin, 0.75% permethrin and 4% DDT were used. Results The major malaria vectors in Tanzania, Anopheles gambiae s.l., were susceptible (mortality rate of 98–100%) to permethrin, deltamethrin, lambdacyhalothrin and DDT in most of the surveyed sites. However, some sites recorded marginal susceptibility (mortality rate of 80–97%); Ilala showed resistance to DDT (mortality rate of 65% [95% CI, 54–74]), and Moshi showed resistance to lambdacyhalothrin (mortality rate of 73% [95% CI, 69–76]) and permethrin (mortality rate of 77% [95% CI, 73–80]). Conclusions The sustained susceptibility of malaria vectors to pyrethroid in Tanzania is encouraging for successful malaria control with Insecticide‐treated nets and IRS. However, the emergency of focal points with insecticide resistance is alarming. Continued monitoring is essential to ensure early containment of resistance, particularly in areas that recorded resistance or marginal susceptibility and those with heavy agricultural and public health use of insecticides.     

First Evidence of High Knockdown Resistance Frequency in Anopheles arabiensis (Diptera: Culicidae) from Ethiopia

The status of knockdown resistance (kdr) mutation was investigated in the major malaria vector Anopheles arabiensis Patton (Diptera: Culicidae) from Ethiopia. Among 240 mosquito samples from 15 villages of southwestern Ethiopia that were screened by allele-specific polymerase chain reaction for kdr mutations, the West African kdr mutation (L1014F) was detected in almost all specimens (98.5%), whereas the East African kdr mutation (L1014S) was absent. Moreover, the mortality of An. gambiae s.l. to diagnostic dosages of 4% DDT, 0.75% permethrin, and 0.05% deltamethrin from bioassay results was 1.0%, 18.1%, and 82.2%, respectively. We report here the highest kdr allele frequency ever observed in An. arabiensis and its implications in malaria vector control in Ethiopia are discussed.     

Insecticide resistance in Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) and Anopheles gambiae Giles (Diptera: Culicidae) could compromise the sustainability of malaria vector control strategies in West Africa

Insecticides from the organophosphate (OP) and pyrethroid (PY) chemical families, have respectively, been in use for 50 and 30 years in West Africa, mainly against agricultural pests, but also against vectors of human disease. The selection pressure, with practically the same molecules year after year (mainly on cotton), has caused insecticide resistance in pest populations such as Bemisia tabaci, vector of harmful phytoviruses on vegetables. The evolution toward insecticide resistance in malaria vectors such as Anopheles gambiae sensus lato (s.l.) is probably related to the current use of these insecticides in agriculture. Thus, successful pest and vector control in West Africa requires an investigation of insect susceptibility, in relation to the identification of species and sub species, such as molecular forms or biotypes. Identification of knock down resistance (kdr) and acetylcholinesterase gene (Ace1) mutations modifying insecticide targets in individual insects and measure of enzymes activity typically involved in insecticide metabolism (oxidase, esterase and glutathion-S-transferase) are indispensable in understanding the mechanisms of resistance. Insecticide resistance is a good example in which genotype–phenotype links have been made successfully. Insecticides used in agriculture continue to select new resistant populations of B. tabaci that could be from different biotype vectors of plant viruses. As well, the evolution of insecticide resistance in An. gambiae threatens the management of malaria vectors in West Africa. It raises the question of priority in the use of insecticides in health and/or agriculture, and more generally, the question of sustainability of crop protection and vector control strategies in the region. Here, we review the susceptibility tests, biochemical and molecular assays data for B. tabaci, a major pest in cotton and vegetable crops, and An. gambiae, main vector of malaria. The data reviewed was collected in Benin and Burkina Faso between 2008 and 2010 under the Corus 6015 research program. This review aims to show: (i) the insecticide resistance in B. tabaci as well as in An. gambiae; and (ii) due to this, the impact of selection of resistant populations on malaria vector control strategies. Some measures that could be beneficial for crop protection and vector control strategies in West Africa are proposed.     

Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel

Pyrethroid insecticides are widely used as one of the most effective control measures in the global fight against agricultural arthropod pests and mosquito-borne diseases, including malaria and dengue. They exert toxic effects by altering the function of voltage-gated sodium channels, which are essential for proper electrical signaling in the nervous system. A major threat to the sustained use of pyrethroids for vector control is the emergence of mosquito resistance to pyrethroids worldwide. Here, we report the successful expression of a sodium channel, AaNa_v1–1, from Aedes aegypti in Xenopus oocytes, and the functional examination of nine sodium channel mutations that are associated with pyrethroid resistance in various Ae. aegypti and Anopheles gambiae populations around the world. Our analysis shows that five of the nine mutations reduce AaNa_v1–1 sensitivity to pyrethroids. Computer modeling and further mutational analysis revealed a surprising finding: Although two of the five confirmed mutations map to a previously proposed pyrethroid-receptor site in the house fly sodium channel, the other three mutations are mapped to a second receptor site. Discovery of this second putative receptor site provides a dual-receptor paradigm that could explain much of the molecular mechanisms of pyrethroid action and resistance as well as the high selectivity of pyrethroids on insect vs. mammalian sodium channels. Results from this study could impact future prediction and monitoring of pyrethroid resistance in mosquitoes and other arthropod pests and disease vectors.Pyrethroids are a large class of synthetic analogs of natural pyrethrins from the flowers of the pyrethrum daisy (Tanacetum cinerariaefolium) (1). They have been used extensively for the control of insect pests and disease vectors for more than three decades. In addition to conventional indoor residue sprays (IRS), use of insecticide-treated nets (ITNs) and long-lasting insecticide-treated nets (LLINs) has been proven to be effective against a wide range of insect vectors involved in the transmission of human diseases, such as malaria, leishmaniasis, Chagas disease, and dengue (2). Currently, ITNs and LLINs are some of the most powerful control measures to reduce malaria morbidity and mortality worldwide. To date, pyrethroids are the only insecticides used for net impregnation in both ITNs and LLIN because of their fast-acting and highly insecticidal activities and their low mammalian toxicity (2, 3).Voltage-gated sodium channels are essential for the initiation and propagation of action potentials in the nervous system and other excitable cells. Like mammalian sodium channels, insect sodium channels comprise four homologous domains (I–IV), each having six membrane spanning segments (S1–S6) (Fig. 1). S4 segments along with S1–S3 segments constitute the voltage-sensing modules, whereas S5, S6, and the membrane-reentrant loop (called the P-region), which connects S5 and S6 segments, form the pore module. In response to membrane depolarization, S4 segments move outward, initiating the opening of sodium channels (i.e., the opening of the activation gate). This process is called activation. According to the crystal structures of the closed potassium channels (4, 5) and the bacterial voltage-gated sodium channel (Na_VAb) (6), the activation gate is located at the cytoplasmic end of the pore, where all four S6 segments converge. After a brief opening, the sodium channel is inactivated. This is achieved by the movement of an inactivation particle (formed mainly by residues in the short intracellular linker connecting domains III and IV), which physically occludes the open pore. Upon repolarization, the sodium channel recovers from the fast inactivation and deactivates (i.e., closing of the activation gate). Pyrethroids mainly inhibit channel deactivation and inactivation, resulting in prolonged opening of sodium channels (7–10). As a consequence, pyrethroids cause repetitive firing and depolarization of the nerve membrane, disrupting the electrical signaling in the insect nervous system (7–9).Open in a separate windowFig. 1.Five pyrethroid-resistance–associated mutations reduce the sensitivity of the AaNa_v1–1 sodium channel to pyrethroids. (A) Topology of the AaNa_v1–1 channel protein illustrating pyrethroid-resistance–associated mutations (solid circles) identified in Aedes aegypti populations around the world and two pyrethroid-resistance–associated mutations identified in Anopheles and Culex species. Sodium channels are large transmembrane proteins with four homologous repeats (I–IV), each having six transmembrane segments (1–6). The mutations are numbered according to amino acid positions in the AaNa_v protein (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU399181","term_id":"214011861","term_text":"EU399181"}}EU399181). S996P, I1018V/M, and V1023G/I correspond to S989P, I1011V/M, and V1016G/I of the house fly sodium channel. (B and C) Channel sensitivity to permethrin (1 μM) (B) and deltamethrin (1 μM) (C). Percentage of channel modification by pyrethroids was determined by the method developed by Tatebayashi et al. (31). The number of oocytes for each mutant construct was >5. Error bars indicate mean ± SEM. Asterisks indicate significant differences from the AaNa_v1–1 channel as determined by using one-way analysis of variance with Scheffé''s post hoc analysis, and significant values were set at P < 0.05.As highlighted recently (11), a major threat to the sustained use of pyrethroids in malaria control is the development of pyrethroid resistance. Indeed, pyrethroid resistance has emerged as one of the most serious concerns to future success of malaria control (references in ref. 12). A prominent mechanism of pyrethroid resistance is called knockdown resistance (kdr), which is caused by mutations in the sodium channel and has been documented globally in almost all major arthropod pests and disease vectors (12–14). To date, more than a dozen kdr mutations have been confirmed to reduce insect sodium channel sensitivity to pyrethroids using the Xenopus oocyte expression system (12–14). Identification of kdr mutations has already led to successful development of rapid and accurate molecular methods to detect pyrethroid resistance in field populations, particularly in various mosquito populations (references in ref. 12).Identification of kdr mutations has also set a foundation for computer modeling and model-guided mutagenesis to address a fundamental question in pyrethroid action and pyrethroid resistance: the elusive pyrethroid-receptor site(s) on the sodium channel. In a previous study, O’Reilly et al. proposed a pyrethroid-receptor site model in the open state of the house fly sodium channel, based on the crystal structure of voltage-gated potassium channels (15). This model predicts that pyrethroids bind to the lipid-exposed interface formed by the linker helix connecting S4 and S5 in domain II (IIL45), and helices IIIS6 and IIS5 (the IIL45–IIS5–IIIS6 triangle model or site 1 hereinafter). Experimental data from systematic site-directed mutagenesis studies in these regions support this model (16, 17). Several key kd mutations in IIL45, IIS5, and IIIS6 are predicted to confer resistance by altering pyrethroid binding (18, 19). However, this model cannot accommodate mutations detected in other regions of the sodium channel, including the L1021F mutation in IIS6 of the mosquito sodium channel. Analogous mutations, e.g., L1014F in the housefly sodium channel and L993F in the cockroach sodium channel are one of the most widespread pyrethroid-resistance mutations in diverse pest species (14, 20). Furthermore, new sodium channel mutations associated with pyrethroid resistance continue to emerge in various arthropod pests, particularly in disease vectors (12). For example, as many as seven new pyrethroid-resistance–associated sodium channel mutations (Fig. 1A) have been reported in Aedes aegypti populations collected from Africa, Asia, and Latin America (12) and, more recently, several new sodium channel mutations are identified to be associated with pyrethroid resistance in Anopheles gambiae (21) and Culex quinquefasciatus (22, 23). Interestingly, many of these mutations are not located in predicted site 1 (i.e., the IIL45–IIS5–IIIS6 triangle) and their role in conferring pyrethroid resistance remains to be demonstrated experimentally.None of pyrethroid-resistance–associated mutations detected in mosquito species has been functionally characterized in mosquito sodium channels due to the lack of a functional expression system for mosquito sodium channels. Several years ago, we initiated an effort to study the molecular action of pyrethroids and pyrethroid resistance in mosquitoes. In this paper, we report the successful expression of the Ae. aegypti sodium channel AaNa_v1–1 in Xenopus oocytes. Establishment of this expression system enabled us to systematically characterize pyrethroid-resistance–associated mutations coupled with computer modeling, which led to the discovery of a second putative pyrethroid-receptor site on mosquito sodium channels.     

Impact of trehalose transporter knockdown on Anopheles gambiae stress adaptation and susceptibility to Plasmodium falciparum infection

Anopheles gambiae is a major vector mosquito for Plasmodium falciparum, the deadly pathogen causing most human malaria in sub-Saharan Africa. Synthesized in the fat body, trehalose is the predominant sugar in mosquito hemolymph. It not only provides energy but also protects the mosquito against desiccation and heat stresses. Trehalose enters the mosquito hemolymph by the trehalose transporter AgTreT1. In adult female A. gambiae, AgTreT1 is predominantly expressed in the fat body. We found that AgTreT1 expression is induced by environmental stresses such as low humidity or elevated temperature. AgTreT1 RNA silencing reduces the hemolymph trehalose concentration by 40%, and the mosquitoes succumb sooner after exposure to desiccation or heat. After an infectious blood meal, AgTreT1 RNA silencing reduces the number of P. falciparum oocysts in the mosquito midgut by over 70% compared with mock-injected mosquitoes. These data reveal important roles for AgTreT1 in stress adaptation and malaria pathogen development in a major vector mosquito. Thus, AgTreT1 may be a potential target for malaria vector control.Critical to the malaria transmission cycle, the mosquito Anopheles gambiae is a major vector for Plasmodium falciparum, the pathogen responsible for most malignant malaria in sub-Saharan Africa. In malaria endemic regions, vector mosquitoes survive harsh fluctuations of temperature and humidity (1). Mosquitoes adapt to environmental changes by adjusting expression levels of certain genes (2); however, most protective mechanisms apparently remain unknown. Recently, we characterized an aquaporin water channel from A. gambiae (AgAQP1) that is important for water homeostasis, because reduced expression protected against dehydration (3). Since water loss has profound effects on mosquito physiology, we investigated other candidate genes that may protect against environmental stress and may affect transmission of P. falciparum.Trehalose is a nonreducing disaccharide of two glucose molecules linked by an α-α-1,1-glycosidic bond. It is abundant in insects, crustaceans, nematodes, bacteria, fungi, and plants, but not vertebrates. As the major sugar in mosquito hemolymph, trehalose is concentrated more than 10 times higher than glucose or other sugars (4). Trehalose is a versatile molecule, serving as the principal energy storage but also as a stabilizer for dry membranes and proteins due to unique chemical and physical properties—high hydration volume, lack of internal hydrogen bonds, and nonreduction (5–8).Trehalose levels rise sharply during several stresses—desiccation (9–12), heat (13), freezing (14, 15), hyperosmolality (16), and oxidation (17). In yeast and plants, trehalose is also a signaling molecule in metabolic pathways affecting growth (18). Evidence is emerging that trehalose protects cultured cells. Increased trehalose in HEK-293 cells expressing Drosophila trehalose-phosphate synthase 1 protects the cells from hypoxic injury (17). Bovine endothelial cell line cultivated with trehalose followed by cryopreservation with trehalose in an optimized solution yielded over 80% viable cells (19). Trehalose levels in anhydrobiotic stage larvae of Polypedilum vanderplanki (sleeping chironomid) accumulate rapidly to ∼20% of the dry body mass, more than five times higher than that of larvae in fresh water (9, 20). Furthermore, a recent study has shown that injection of d-(+)-trehalose into the hemocoel of head-intact, starved cockroaches lowers the content of short neuropeptide F in hemolymph, suggesting novel roles of trehalose in regulating brain and midgut interplay in insect digestion and nutrition-associated behavior (21).Synthesized exclusively in the fat body of mosquitoes, trehalose is transported to the circulating hemolymph for delivery to other tissues. This process involves the specific movement of trehalose across cell membranes facilitated by the trehalose transporter, TreT (9, 22). The AgTreT1 cDNA from A. gambiae is an ortholog of PvTreT1 from P. vanderplanki. Only one TreT gene is present in the A. gambiae genome, and its trehalose-transport function was characterized by heterologous expression in Xenopus oocytes (22). PvTreT1 was proposed to contribute to the dehydration resistance of P. vanderplanki larvae in vivo (9). Nevertheless, no direct evidence has supported this role of AgTreT1 in the whole vector mosquito A. gambiae.Trehalose is a likely energy source for Plasmodium pathogens in A. gambiae mosquitoes. After ingesting an infected blood meal, Plasmodium gametocytes differentiate into male or female gametes and fuse to form ookinetes in the mosquito midgut. Mobile ookinetes then penetrate the gut lining to produce oocysts on the basal-lamina side. During the oocyst stage, malaria parasites amplify by several thousand fold, scavenging energy from the vector. Plasmodium infection has been reported to deplete sugars in vector hemolymph, suggesting that trehalose is used by parasites for rapid growth (23). Genes related to trehalose transport and metabolism may be related to the life cycle of Plasmodium spp. in mosquito vectors.In this study, we observed that AgTreT1 expression is induced by desiccation and heat. Reducing AgTreT1 expression in female A. gambiae by RNAi decreases hemolymph trehalose levels. Mosquitoes with reduced AgTreT1 levels die sooner than controls in dry or hot environments. Moreover, when AgTreT1 was silenced in A. gambiae infected with P. falciparum, significantly fewer parasite oocysts appear in the midguts than in midguts of control mosquitoes. These data suggest an important role of AgTreT1 in maintaining vector hemolymph sugar levels during desiccation and heat and reveal a unique function in P. falciparum propagation during the oocyst stage.     

A significant increase in kdr in Anopheles gambiae is associated with an intensive vector control intervention in Burundi highlands

Objectives and Methods In Burundi, the occurrence of the knock down resistance (kdr) mutation in Anopheles gambiae sensu lato (s.l.) was determined for six consecutive years within the framework of a vector control programme. Findings were also linked with the insecticide resistance status observed with bioassay in An. gambiae s.l. and An. funestus. Results The proportion of An. gambiae s.l. carrying the East Leu‐Ser kdr mutation was 1% before the spraying intervention in 2002; by 2007 it was 86% in sprayed valleys and 67% in untreated valleys. Multivariate analysis showed that increased risk of carrying the kdr mutation is associated with spraying interventions, location and time. In bioassays conducted between 2005 and 2007 at five sites, An. funestus was susceptible to permethrin, deltamethrin and DDT. Anopheles gambiae s.l. remained susceptible or tolerant to deltamethrin and resistant to DDT and permethrin, but only when kdr allele carriers reached 90% of the population. Conclusions The cross‐resistance against DDT and permethrin in Karuzi suggests a possible kdr resistance mechanism. Nevertheless, the homozygous resistant genotype alone does not entirely explain the bioassay results, and other mechanisms conferring resistance cannot be ruled out. After exposure to all three insecticides, homozygote individuals for the kdr allele dominate among the surviving An. gambiae s.l. This confirms the potential selection pressure of pyrethroids on kdr mutation. However, the high occurrence of the kdr mutation, even at sites far from the sprayed areas, suggests a selection pressure other than that exerted by the vector control programme.     

Plasmodium evasion of mosquito immunity and global malaria transmission: The lock-and-key theory

Plasmodium falciparum malaria originated in Africa and became global as humans migrated to other continents. During this journey, parasites encountered new mosquito species, some of them evolutionarily distant from African vectors. We have previously shown that the Pfs47 protein allows the parasite to evade the mosquito immune system of Anopheles gambiae mosquitoes. Here, we investigated the role of Pfs47-mediated immune evasion in the adaptation of P. falciparum to evolutionarily distant mosquito species. We found that P. falciparum isolates from Africa, Asia, or the Americas have low compatibility to malaria vectors from a different continent, an effect that is mediated by the mosquito immune system. We identified 42 different haplotypes of Pfs47 that have a strong geographic population structure and much lower haplotype diversity outside Africa. Replacement of the Pfs47 haplotypes in a P. falciparum isolate is sufficient to make it compatible to a different mosquito species. Those parasites that express a Pfs47 haplotype compatible with a given vector evade antiplasmodial immunity and survive. We propose that Pfs47-mediated immune evasion has been critical for the globalization of P. falciparum malaria as parasites adapted to new vector species. Our findings predict that this ongoing selective force by the mosquito immune system could influence the dispersal of Plasmodium genetic traits and point to Pfs47 as a potential target to block malaria transmission. A new model, the “lock-and-key theory” of P. falciparum globalization, is proposed, and its implications are discussed.The most deadly form of malaria in humans is caused by Plasmodium falciparum parasites. Malaria originated in Africa (1, 2) and is transmitted by anopheline mosquitoes. The disease became global as humans migrated to other continents and parasites encountered different mosquito species that were sometimes evolutionarily distant from African vectors (3). For example, anophelines of the subgenus Nyssorhynchus (malaria vectors in Central and South America, such as Anopheles albimanus) diverged from the subgenus Cellia (malaria vectors in Africa, India, and South Asia) about 100 Mya (4). P. falciparum parasites are transmitted by more than 70 different anopheline species worldwide (3), but compatibilities differ between specific vector–parasite combinations (5). For example, P. falciparum NF54 (Pf NF54), of putative African origin, effectively infects Anopheles gambiae, the main malaria vector in sub-Saharan Africa; but A. albimanus is highly refractory to this strain (6–8); whereas Asian P. falciparum isolates infect Anopheles stephensi (Nijmegen strain), a major vector in India, more effectively than A. gambiae (9). Similar differences in compatibility have been reported between Plasmodium vivax and different anopheline species (10, 11). The A. gambiae immune system can mount effective antiplasmodial responses mediated by the complement-like system that limit infection (12). We have previously shown that some P. falciparum lines can avoid detection by the A. gambiae immune system (13) and identified Pfs47 as the gene that mediated immune evasion (14). Here, we present direct evidence of selection of P. falciparum by the mosquito immune system and show that providing P. falciparum with a Pfs47 haplotype compatible for a given anopheline mosquito is sufficient for the parasite to evade mosquito immunity. The implications of P. falciparum selection by mosquitoes for global malaria transmission are discussed.     

Knockdown resistance mutations (kdr) and insecticide susceptibility to DDT and pyrethroids in Anopheles gambiae from Equatorial Guinea

Objectives To determine the frequency of knockdown resistance (kdr) mutations in the malaria vector Anopheles gambiae s.s. from continental Equatorial Guinea; and to relate kdr genotypes with susceptibility to DDT and pyrethroid insecticides in this vector. Methods Female mosquitoes were collected in two villages, Miyobo and Ngonamanga, of mainland Equatorial Guinea. Insecticide susceptibility tests were performed following WHO procedures. Anopheles gambiae complex specimens were identified to species and molecular form by PCR. Genotyping of the kdr locus was performed by allele‐specific PCR and direct sequencing in a subset of samples. Results Both M and S molecular forms of A. gambiae were found in Ngonamanga whereas only the S‐form was identified in Miyobo. The two kdr mutations were detected in S‐form samples of both villages, with a higher frequency of the kdr‐e (Leu‐1014‐Ser) allele (Miyobo: 16%; Ngonamanga: 40%). The kdr‐w (Leu‐1014‐Phe) mutation was also detected in 3% of the M‐form. All individuals tested for pyrethroids were susceptible. A mortality rate of 86% was obtained for DDT. An overall kdr allele frequency (i.e. kdr‐e + kdr‐w) of 22% was detected in DDT resistant individuals, whereas susceptible individuals had a kdr frequency of 6%. Conclusion The co‐occurrence of both kdr mutations and reduced susceptibility to DDT found in A. gambiae highlights the importance of implementing efficient surveillance of insecticide resistance in Equatorial Guinea.     

Susceptibility of Anopheles gambiae s.l. to DDT, malathion, permethrin and deltamethrin in Ethiopia

Objective To assess the susceptibility/resistance level of Anopheles gambiae s.l. to DDT, malathion, permethrin and deltamethrin in different parts of Ethiopia. Methods Field collected female An.gambiae s.l. was exposed for 1 h to discriminating dosage of 4% DDT, 5% malathion, 0.75% permethrin and 0.05% deltamethrin using WHO insecticide susceptibility test kits and procedures. Knockdown and mortality rates were recorded at 10, 15, 20, 30, 40, 50 and 60 min and 24 h post‐exposure respectively. Results Anopheles gambiae s.l. was sensitive to DDT only in 2 of 16 localities where susceptibility studies were carried out in northern Ethiopia; it was resistant in 11 sites and potentially resistant in three. To malathion, the test population was sensitive in four of the six study sites in southern Ethiopia and potentially resistant in the other two sites. In northern Ethiopia, the population was resistant in five localities and sensitive in three. Of the six localities in northern Ethiopia where permethrin was tested, populations were sensitive in three, resistant in one and potentially resistant in two. In southern Ethiopia, the populations were resistant in five of the six sites. Against deltamethrin, the population was sensitive in five of 13 localities, three in northern and two in southern Ethiopia. It was resistant only in two localities, one in northern and one in southern Ethiopia, and potentially resistant in five localities. In eastern Ethiopia at Sabure, the population was sensitive to all insecticides but DDT to which it was potentially resistant. Conclusion The existence of high level of DDT and pyrethroid resistance with the possibility of cross‐resistance to each other and other classes of agricultural pesticides could seriously jeopardise the efficacy of both ITNs and IRS in the country in the future. Insecticide resistance monitoring and surveillance systems as part of a malaria control programme are mandatory for proper management of resistance. The use of a mixture of unrelated insecticides for impregnating nets and rotational use of insecticides for IRS is suggested as a way forward.     

Site-specific genetic engineering of the Anopheles gambiae Y chromosome

Despite its function in sex determination and its role in driving genome evolution, the Y chromosome remains poorly understood in most species. Y chromosomes are gene-poor, repeat-rich and largely heterochromatic and therefore represent a difficult target for genetic engineering. The Y chromosome of the human malaria vector Anopheles gambiae appears to be involved in sex determination although very little is known about both its structure and function. Here, we characterize a transgenic strain of this mosquito species, obtained by transposon-mediated integration of a transgene construct onto the Y chromosome. Using meganuclease-induced homologous repair we introduce a site-specific recombination signal onto the Y chromosome and show that the resulting docking line can be used for secondary integration. To demonstrate its utility, we study the activity of a germ-line–specific promoter when located on the Y chromosome. We also show that Y-linked fluorescent transgenes allow automated sex separation of this important vector species, providing the means to generate large single-sex populations. Our findings will aid studies of sex chromosome function and enable the development of male-exclusive genetic traits for vector control.Mosquito species of the Anopheles gambiae complex represent the principal vectors of human malaria, and they pose an enormous burden on global health and economies. Every year, 300–500 million people are infected by malaria and more than 1 million people die as a consequence of Plasmodium parasite infections (1). The malaria mosquito A. gambiae has two pairs of autosomes, termed 2 and 3, and a pair of heteromorphic sex chromosomes X and Y, XX in females and XY in males (2). Extensive nonpairing regions exist between the X and the degenerate Y chromosome. and evidence points to a factor located on the Y chromosome that primarily determines the sex in Anopheles (3). Current models suggest that the evolutionary differentiation of Y chromosomes begins with the acquisition of a male determining factor on a proto-Y chromosome (4, 5). This event is followed by a progressive suppression of recombination between the still largely homomorphic proto-sex chromosomes, a process attributed to the acquisition of sexually antagonistic mutations, which are beneficial to the heterogametic sex but detrimental to the homogametic sex (6–8). The lack of recombination, together with the male-limited transmission, leads to the degeneration of the Y chromosome, which involves accumulation of deleterious mutations, spread of transposable elements, and silencing of all or most of the genes present on the proto-Y (9–11). As a result, Y chromosomes of many species appear to be strongly heterochromatic and harbor only few genes often involved in male fertility (12–17). The accumulation of repetitive sequences, many of which are also present on other chromosomes, hampers the assembly of Y chromosome contigs following shotgun sequencing. Indeed, despite the completion of the A. gambiae genome project (18), and the knowledge that the primary signal is likely to be associated with the inheritance of the Y (3, 19), no assembly of the Anopheles Y chromosome has been achieved. At present, public databases host only a few hundred kilobases of A. gambiae sequences attributed to the Y, a chromosome that is estimated to comprise 10% of the genome and to be at least 20 Mb in size. None of these Y-specific scaffolds have been physically mapped, because the Y chromosome does not polytenize. The exploration of the Y chromosome will improve our understanding of the evolutionary forces involved in driving chromosome evolution and may enable the manipulation of the molecular pathways that control sex determination and sexual differentiation in mosquitoes. In a number of organisms, Y chromosome genes have been found to be essential for male fertility or sex determination. Recently, a number of excellent candidate genes potentially involved in these processes have been identified on the Y chromosome of anopheline mosquitoes (20, 21). Because interfering with male fertility is an essential part of vector control strategies such as the sterile insect technique, the identification of such genes is of particular interest to mosquito biologists. In this paper, we demonstrate the targeted molecular manipulation of the Y chromosome in A. gambiae, thus opening up a number of ways to explore one of the most fascinating of evolution’s upshots and to harness this genetic tool for vector control.     

Vectored antibody gene delivery protects against Plasmodium falciparum sporozoite challenge in mice

Malaria caused by Plasmodium falciparum kills nearly one million children each year and imposes crippling economic burdens on families and nations worldwide. No licensed vaccine exists, but infection can be prevented by antibodies against the circumsporozoite protein (CSP), the major surface protein of sporozoites, the form of the parasite injected by mosquitoes. We have used vectored immunoprophylaxis (VIP), an adeno-associated virus-based technology, to introduce preformed antibody genes encoding anti-P. falciparum CSP mAb into mice. VIP vector-transduced mice exhibited long-lived mAb expression at up to 1,200 µg/mL in serum, and up to 70% were protected from both i.v. and mosquito bite challenge with transgenic Plasmodium berghei rodent sporozoites that incorporate the P. falciparum target of the mAb in their CSP. Serum antibody levels and protection from mosquito bite challenge were dependent on the dose of the VIP vector. All individual mice expressing CSP-specific mAb 2A10 at 1 mg/mL or more were completely protected, suggesting that in this model system, exceeding that threshold results in consistent sterile protection. Our results demonstrate the potential of VIP as a path toward the elusive goal of immunization against malaria.Among infectious diseases, malaria ranks fourth as a cause of death. In Africa in 2012, 500,000–800,000 deaths, mostly among children under 5 y of age, resulted from ∼200 million clinical cases of malaria caused by Plasmodium falciparum, the parasite species responsible for most malaria mortality (1). In addition to its direct effect on health, malaria imposes severe economic burdens. These include crippling treatment costs at the level of individual families and a significant contribution to low national incomes and reduced overall rates of economic growth in nations worldwide (2, 3). The human and economic burdens imposed by malaria make malaria reduction a critical global priority.Traditional approaches to malaria control such as antimalarial drug treatment, mosquito control by habitat modification and insecticide use, and reduction of exposure to infected mosquitoes have had substantial success in reducing malaria incidence and malaria-specific mortality rates (4). However, these achievements may be difficult to sustain in the face of growing drug resistance among parasites, insecticide resistance in vector populations, and the reduced funding for malaria control anticipated by the World Health Organization for the near future (4). If continued progress against malaria is to be made, it is essential that new approaches for malaria prevention be added to currently available tools.Sporozoites are the infectious form of the malaria parasite injected by Anopheles mosquitoes. It has been known for decades that immunization of animals or humans with radiation-attenuated sporozoites can elicit sterilizing immunity to malaria, preventing infection, pathogenesis, and transmission (5–8). The predominant antibody response to immunization by irradiated sporozoites is to the circumsporozoite protein (CSP), which coats the sporozoite surface (9, 10). In in vivo and in vitro models of P. falciparum sporozoite infection, infection can be blocked completely by antibody to the immunodominant epitope of CSP, a tetrapeptide [asn-ala-asn-pro (NANP)] found in 30 or more tandemly repeated copies in the central region of the protein (11–13). The NANP repeat is stringently conserved in P. falciparum isolates from diverse geographical locations (14) and thus represents a potentially universal target for P. falciparum immunity. The most advanced malaria infection-blocking vaccine candidate, RTS,S, is comprised of hepatitis B virus-like particles that display a carboxyl-terminal segment of P. falciparum CSP, including part of the central NANP repeat region. Immunization with RTS,S reduces incidence of clinical malaria by 40–70% in children, but levels of protection wane with time (15–18).Vectored immunoprophylaxis (VIP) provides an alternative to conventional immunization as a route to protective antibody expression (19–21). VIP employs optimized adeno-associated virus (AAV) based vectors to deliver genes encoding mAb with previously characterized specificities to animals. Intramuscular injection of VIP vectors in mice and macaques elicits long-lived antibody or antibody-related immunoadhesin production at levels sufficient to protect against HIV, simian immunodeficiency virus, and influenza A virus infection (19–22). Here we report that in mice, VIP-directed production of sporozoite-neutralizing mAb against the P. falciparum CSP central repeat can confer sterile immunity to infection by a transgenic strain of the rodent parasite Plasmodium berghei whose CSP contains the P. falciparum CSP central repeat [Pb/Pf (12)].