Comparative analysis of the Potter Tower and a new Track Sprayer for the application of residual sprays in the laboratory.

BACKGROUND: Efforts to evaluate the residual efficacy of new indoor residual spraying (IRS) formulations have identified limitations with the industry standard laboratory sprayer, the Potter Spray Tower (PT). Calibrating the PT can be time-consuming, and the dosing of surfaces may not be as accurate or uniform as previously assumed. METHODS: To address these limitations, the Micron Horizontal Track Sprayer with Spray Cabinet (TS) was developed to provide higher efficiency, ease of operation and deposition uniformity equal to or better than the PT. A series of studies were performed using a fluorescent tracer and three IRS formulations (Actellic® 300CS, K-Othrine WG250 and Suspend PolyZone) sprayed onto surfaces using either the PT or the TS. RESULTS: Deposition volumes could be accurately calibrated for both spray systems. However, the uniformity of spray deposits was higher for the TS compared to the PT. Less than 12% of the volume sprayed using the PT reaches the target surface, with the remaining 88% unaccounted for, presumably vented out of the fume hood or coating the internal surfaces of the tower. In contrast, the TS deposits most of the spray on the floor of the spray chamber, with the rest contained therein. The total sprayed surface area in one run of the TS is 1.2 m2, and the operational zone for spray target placement is 0.7 m2, meaning that 58% of the applied volume deposits onto the targets. The TS can treat multiple surfaces (18 standard 15 × 15 cm tiles) in a single application, whereas the PT treats one surface at a time and a maximum area of around 0.0225 m2. An assessment of the time taken to perform spraying, including the setup, calibration and cleaning, showed that the cost of application using the TS was around 25-35 × less per tile sprayed. Standard operating procedures (SOPs) for calibration and use of both the Potter Tower and Track Sprayer have been developed. CONCLUSIONS: Overall, the TS represents a significant improvement over the PT in terms of the efficiency and accuracy of IRS formulation applications onto test substrates and offers a useful additional tool for researchers and manufacturers wanting to screen new active ingredients or evaluate the efficacy of IRS or other sprayable formulations for insect control.

Community evaluation of VECTRON™ T500, a broflanilide insecticide, for indoor residual spraying for malaria vector control in central Benin; a two arm non-inferiority cluster randomised trial.

VECTRON™ T500 is a wettable powder IRS formulation of broflanilide, a newly discovered insecticide. We performed a two-arm non-inferiority community randomised evaluation of VECTRON™ T500, compared to Fludora® Fusion against pyrethroid-resistant Anopheles gambiae s.l. in an area of high coverage with pyrethroid-only nets in the Za-Kpota District of central Benin. One round of IRS was applied in all consenting households in the study area. Sixteen clusters were randomised (1:1) to receive VECTRON™ T500 (100 mg/m2 for broflanilide) or Fludora® Fusion (200 mg/m2 for clothianidin and 25 mg/m2 for deltamethrin). Surveys were performed to assess adverse events and the operational feasibility and acceptability of VECTRON™ T500 among spray operators and household inhabitants. Human landing catches were conducted in 6 households every 1-2 months for up to 18 months post-intervention to assess the impact on vector densities, sporozoite rates and entomological inoculation rates. Bottle bioassays were performed to monitor vector susceptibility to pyrethroids, broflanilide and clothianidin. Monthly wall cone bioassays were conducted for 24 months to assess the residual efficacy of the IRS formulations using susceptible and pyrethroid-resistant An. gambiae s.l. A total of 26,562 female mosquitoes were collected during the study, of which 40% were An. gambiae s.l., the main malaria vector in the study area. The vector population showed high intensity pyrethroid resistance but was susceptible to broflanilide (6 µg/bottle) and clothianidin (90 µg/bottle). Using a non-inferiority margin of 50%, vector density indicated by the human biting rate (bites/person/night) was non-inferior in the VECTRON™ T500 arm compared to the Fludora® Fusion arm both indoors (0.846 bites/p/n in Fludora® Fusion arm vs. 0.741 bites/p/n in VECTRON™ T500 arm, IRR 0.54, 95% CI 0.22-1.35, p = 0.150) and outdoors (0.691 bites/p/n in Fludora® Fusion arm vs. 0.590 bites/p/n in VECTRON™ T500 clusters, IRR 0.75, 95% CI 0.41-1.38, p = 0.297). Sporozoite rates and entomological inoculation rates did not differ significantly between study arms (sporozoite rate: 0.9% vs 1.1%, p = 0. 0.746, EIR: 0.008 vs 0.006 infective bites per person per night, p = 0.589). Cone bioassay mortality with both VECTRON™ T500 and Fludora® Fusion was 100% for 24 months post-IRS application on both cement and mud treated house walls with both susceptible and pyrethroid-resistant strains of An. gambiae s.l. Perceived adverse events reported by spray operators and householders were generally very low (< 6%) in both study arms. VECTRON™ T500 was non-inferior to Fludora® Fusion in reducing the risk of malaria transmission by pyrethroid resistant vectors when applied for IRS in communities in central Benin. The insecticide showed prolonged residual efficacy on house walls, lasting over 24 months and had a high acceptability with homeowners. Community application of VECTRON™ T500 for IRS provides improved and prolonged control of pyrethroid resistant malaria vectors and enhances our capacity to manage insecticide resistance.

VECTRON™ T500, a new broflanilide insecticide for indoor residual spraying, provides prolonged control of pyrethroid-resistant malaria vectors.

BACKGROUND: Broflanilide is a newly discovered insecticide with a novel mode of action targeting insect γ-aminobutyric acid receptors. The efficacy of VECTRON™ T500, a wettable powder formulation of broflanilide, was assessed for IRS against wild pyrethroid-resistant malaria vectors in experimental huts in Benin. METHODS: VECTRON™ T500 was evaluated at 100 mg/m2 in mud and cement-walled experimental huts against wild pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) in Covè, southern Benin, over 18 months. A direct comparison was made with Actellic® 300CS, a WHO-recommended micro-encapsulated formulation of pirimiphos-methyl, applied at 1000 mg/m2. The vector population at Covè was investigated for susceptibility to broflanilide and other classes of insecticides used for vector control. Monthly wall cone bioassays were performed to assess the residual efficacy of VECTRON™ T500 using insecticide susceptible An. gambiae Kisumu and pyrethroid-resistant An. gambiae s.l. Covè strains. The study complied with OECD principles of good laboratory practice. RESULTS: The vector population at Covè was resistant to pyrethroids and organochlorines but susceptible to broflanilide and pirimiphos-methyl. A total of 23,171 free-flying wild pyrethroid-resistant female An. gambiae s.l. were collected in the experimental huts over 12 months. VECTRON™ T500 induced 56%-60% mortality in wild vector mosquitoes in both cement and mud-walled huts. Mortality with VECTRON™ T500 was 62%-73% in the first three months and remained > 50% for 9 months on both substrate-types. By comparison, mortality with Actellic® 300CS was very high in the first three months (72%-95%) but declined sharply to < 40% after 4 months. Using a non-inferiority margin defined by the World Health Organization, overall mortality achieved with VECTRON™ T500 was non-inferior to that observed in huts treated with Actellic® 300CS with both cement and mud wall substrates. Monthly in situ wall cone bioassay mortality with VECTRON™ T500 also remained over 80% for 18 months but dropped below 80% with Actellic® 300CS at 6-7 months post spraying. CONCLUSION: VECTRON™ T500 shows potential to provide substantial and prolonged control of malaria transmitted by pyrethroid-resistant mosquito vectors when applied for IRS. Its addition to the current list of WHO-approved IRS insecticides will provide a suitable option to facilitate rotation of IRS products with different modes of action.

Efficacy of bednets with dual insecticide-treated netting (Interceptor® G2) on side and roof panels against Anopheles arabiensis in north-eastern Tanzania.

BACKGROUND: Optimising insecticide use and managing insecticide resistance are important to sustain gains against malaria using long-lasting insecticidal nets (LLINs). Restricting insecticides to where mosquitoes are most likely to make multiple contacts could reduce the quantity of insecticide needed to treat the nets. Previous studies have shown that nets partially treated with a pyrethroid insecticide had equivalent mortality compared to a fully treated net. This study compared the efficacy of: (i) whole Interceptor® G2 nets (IG2; a dual-active LLIN containing alpha-cypermethrin and chlorfenapyr), (ii) nets with roof panels made of IG2 netting, (iii) nets with side panels made of IG2 netting and (iv) whole untreated nets as test nets. METHODS: The study was conducted in cow-baited experimental huts, Moshi Tanzania, using a four-arm Latin square design. Test nets had 30 holes cut in panels to simulate a typical net after 2-3 year use. The trial data were analysed using generalized linear models with mortality, blood-feeding, exophily and deterrence against wild free-flying Anopheles arabiensis as outcomes and test nets as predictors. RESULTS: Mortality was significantly higher in the nets with roof IG2 [27%, P = 0.001, odds ratio (OR) = 51.0, 95% CI = 4.8-546.2), side IG2 (44%, P < 0.001, OR = 137.6, 95% CI = 12.2-1553.2] and whole IG2 (53%, P < 0.001, OR = 223.0, 95% CI = 19.07-2606.0) nettings than the untreated (1%) nets. Mortality was also significantly higher in the whole IG2 net compared to the net with roof IG2 netting (P = 0.009, OR = 4.4, 95% CI = 1.4-13.3). Blood feeding was 22% in untreated, 10% in roof IG2, 14% in side IG2 and 19% in whole IG2 nets. Exiting was 92% in untreated, 89% in roof IG2, 97% in side IG2 and 94% whole IG2 nets. CONCLUSION: The results show that although the roof-treated IG2 net induced greater mortality compared to untreated nets, its efficacy was reduced compared to whole IG2 nets. Therefore, there was no benefit to be gained from restricting dual-active ingredient IG2 netting to the roof of nets.

Assessment of the Impact of Potential Tetracycline Exposure on the Phenotype of Aedes aegypti OX513A: Implications for Field Use.

BACKGROUND: Aedes aegypti is the primary vector of dengue fever, a viral disease which has an estimated incidence of 390 million infections annually. Conventional vector control methods have been unable to curb the transmission of the disease. We have previously reported a novel method of vector control using a tetracycline repressible self-limiting strain of Ae. aegypti OX513A which has achieved >90% suppression of wild populations. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the impact of tetracycline and its analogues on the phenotype of OX513A from the perspective of possible routes and levels of environmental exposure. We determined the minimum concentration of tetracycline and its analogues that will allow an increased survivorship and found these to be greater than the maximum concentration of tetracyclines found in known Ae. aegypti breeding sites and their surrounding areas. Furthermore, we determined that OX513A parents fed tetracycline are unable to pre-load their progeny with sufficient antidote to increase their survivorship. Finally, we studied the changes in concentration of tetracycline in the mass production rearing water of OX513A and the developing insect. CONCLUSION/SIGNIFICANCE: Together, these studies demonstrate that potential routes of exposure of OX513A individuals to tetracycline and its analogues in the environment are not expected to increase the survivorship of OX513A.

Mating competitiveness and life-table comparisons between transgenic and Indian wild-type Aedes aegypti L.

BACKGROUND: OX513A is a genetically engineered strain of Aedes aegypti carrying a repressible, dominantly inherited transgene that confers lethality in immature heterozygous progeny. Released male OX513A adults have proven to be effective for the localised suppression of wild Ae. aegypti, highlighting its potential in vector control. Mating and life-table assessments were used to compare OX513A with reared Ae. aegypti strains collected from New Delhi and Aurangabad regions in India. RESULTS: Mating proportions of New Delhi females versus males of OX513A or New Delhi strains were 0.52 and 0.48 respectively, indicating no discrimination by females against either strain, and males of both strains were equally competitive. Developmental time from first instar to adult emergence was significantly longer for OX513A (10.7 ± 0.04 days) than for New Delhi (9.4 ± 0.04 days) and Aurangabad strains (9.1 ± 0.04 days). Differences in mean longevities, female reproductive parameters and population growth parameters between the strains were non-significant. CONCLUSIONS: The laboratory study demonstrates that only minor life-table variations of limited biological relevance exist between OX513A and Indian Ae. aegypti populations, and males had equal potential for mating competitiveness. Thus, results support the OX513A strain as a suitable candidate for continued evaluation towards sustainable management of Ae. aegypti populations in India.

Estimation of Aedes aegypti (Diptera: Culicidae) population size and adult male survival in an urban area in Panama.

Traditional mosquito control strategies rely heavily on the use of chemical insecticides. However, concerns about the efficiency of traditional control methods, environmental impact and emerging pesticide resistance have highlighted the necessity for developing innovative tools for mosquito control. Some novel strategies, including release of insects carrying a dominant lethal gene (RIDL®), rely on the sustained release of modified male mosquitoes and therefore benefit from a thorough understanding of the biology of the male of the species. In this report we present the results of a mark-release-recapture study aimed at: (i) establishing the survival in the field of laboratory-reared, wild-type male Aedes aegypti and (b) estimating the size of the local adult Ae. aegypti population. The study took place in Panama, a country where recent increases in the incidence and severity of dengue cases have prompted health authorities to evaluate alternative strategies for vector control. Results suggest a life expectancy of 2.3 days for released male mosquitoes (confidence interval: 1.78-2.86). Overall, the male mosquito population was estimated at 58 males/ha (range 12-81 males/ha), which can be extrapolated to an average of 0.64 pupae/person for the study area. The practical implications of these results are discussed.

Estimation of Aedes aegypti (Diptera: Culicidae) population size and adult male survival in an urban area in Panama

Traditional mosquito control strategies rely heavily on the use of chemical insecticides. However, concerns about the efficiency of traditional control methods, environmental impact and emerging pesticide resistance have highlighted the necessity for developing innovative tools for mosquito control. Some novel strategies, including release of insects carrying a dominant lethal gene (RIDL®), rely on the sustained release of modified male mosquitoes and therefore benefit from a thorough understanding of the biology of the male of the species. In this report we present the results of a mark-release-recapture study aimed at: (i) establishing the survival in the field of laboratory-reared, wild-type male Aedes aegypti and (b) estimating the size of the local adult Ae. aegypti population. The study took place in Panama, a country where recent increases in the incidence and severity of dengue cases have prompted health authorities to evaluate alternative strategies for vector control. Results suggest a life expectancy of 2.3 days for released male mosquitoes (confidence interval: 1.78-2.86). Overall, the male mosquito population was estimated at 58 males/ha (range 12-81 males/ha), which can be extrapolated to an average of 0.64 pupae/person for the study area. The practical implications of these results are discussed.

Mass production of genetically modified Aedes aegypti for field releases in Brazil.

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)(1-3). A control method known as RIDL (Release of Insects carrying a Dominant Lethal)(4), is based around SIT, but uses genetic methods to remove the need for radiation-sterilization(5-8). A RIDL strain of Ae. aegypti was successfully tested in the field in Grand Cayman(9,10); further field use is planned or in progress in other countries around the world. Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL control program, released as either pupae or adults(11,12). To suppress a mosquito population using RIDL a large number of high quality male adults need to be reared(13,14). The following describes the methods for the mass rearing of OX513A, a RIDL strain of Ae. aegypti (8), for release and covers the techniques required for the production of eggs and mass rearing RIDL males for a control program.

Genetic control of Aedes mosquitoes.

Aedes mosquitoes include important vector species such as Aedes aegypti, the major vector of dengue. Genetic control methods are being developed for several of these species, stimulated by an urgent need owing to the poor effectiveness of current methods combined with an increase in chemical pesticide resistance. In this review we discuss the various genetic strategies that have been proposed, their present status, and future prospects. We focus particularly on those methods that are already being tested in the field, including RIDL and Wolbachia-based approaches.

Fitness of transgenic mosquito Aedes aegypti males carrying a dominant lethal genetic system.

OX513A is a transgenic strain of Aedes aegypti engineered to carry a dominant, non-sex-specific, late-acting lethal genetic system that is repressed in the presence of tetracycline. It was designed for use in a sterile-insect (SIT) pest control system called RIDL® (Release of Insects carrying a Dominant Lethal gene) by which transgenic males are released in the field to mate with wild females; in the absence of tetracycline, the progeny from such matings will not survive. We investigated the mating fitness of OX513A in the laboratory. Male OX513A were as effective as Rockefeller (ROCK) males at inducing refractoriness to further mating in wild type females and there was no reduction in their ability to inseminate multiple females. They had a lower mating success but yielded more progeny than the wild-type comparator strain (ROCK) when one male of each strain was caged with a ROCK female. Mating success and fertility of groups of 10 males-with different ratios of RIDL to ROCK-competing for five ROCK females was similar, but the median longevity of RIDL males was somewhat (18%) lower. We conclude that the fitness under laboratory conditions of OX513A males carrying a tetracycline repressible lethal gene is comparable to that of males of the wild-type comparator strain.

Oral ingestion of transgenic RIDL Ae. aegypti larvae has no negative effect on two predator Toxorhynchites species.

Dengue is the most important mosquito-borne viral disease. No specific treatment or vaccine is currently available; traditional vector control methods can rarely achieve adequate control. Recently, the RIDL (Release of Insect carrying Dominant Lethality) approach has been developed, based on the sterile insect technique, in which genetically engineered 'sterile' homozygous RIDL male insects are released to mate wild females; the offspring inherit a copy of the RIDL construct and die. A RIDL strain of the dengue mosquito, Aedes aegypti, OX513A, expresses a fluorescent marker gene for identification (DsRed2) and a protein (tTAV) that causes the offspring to die. We examined whether these proteins could adversely affect predators that may feed on the insect. Aedes aegypti is a peri-domestic mosquito that typically breeds in small, rain-water-filled containers and has no specific predators. Toxorhynchites larvae feed on small aquatic organisms and are easily reared in the laboratory where they can be fed exclusively on mosquito larvae. To evaluate the effect of a predator feeding on a diet of RIDL insects, OX513A Ae. aegypti larvae were fed to two different species of Toxorhynchites (Tx. splendens and Tx. amboinensis) and effects on life table parameters of all life stages were compared to being fed on wild type larvae. No significant negative effect was observed on any life table parameter studied; this outcome and the benign nature of the expressed proteins (tTAV and DsRed2) indicate that Ae. aegypti OX513A RIDL strain is unlikely to have any adverse effects on predators in the environment.

The orthologue of the fruitfly sex behaviour gene fruitless in the mosquito Aedes aegypti: evolution of genomic organisation and alternative splicing.

In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control.

Open field release of genetically engineered sterile male Aedes aegypti in Malaysia.

BACKGROUND: Dengue is the most important mosquito-borne viral disease. In the absence of specific drugs or vaccines, control focuses on suppressing the principal mosquito vector, Aedes aegypti, yet current methods have not proven adequate to control the disease. New methods are therefore urgently needed, for example genetics-based sterile-male-release methods. However, this requires that lab-reared, modified mosquitoes be able to survive and disperse adequately in the field. METHODOLOGY/PRINCIPAL FINDINGS: Adult male mosquitoes were released into an uninhabited forested area of Pahang, Malaysia. Their survival and dispersal was assessed by use of a network of traps. Two strains were used, an engineered 'genetically sterile' (OX513A) and a wild-type laboratory strain, to give both absolute and relative data about the performance of the modified mosquitoes. The two strains had similar maximum dispersal distances (220 m), but mean distance travelled of the OX513A strain was lower (52 vs. 100 m). Life expectancy was similar (2.0 vs. 2.2 days). Recapture rates were high for both strains, possibly because of the uninhabited nature of the site. CONCLUSIONS/SIGNIFICANCE: After extensive contained studies and regulatory scrutiny, a field release of engineered mosquitoes was safely and successfully conducted in Malaysia. The engineered strain showed similar field longevity to an unmodified counterpart, though in this setting dispersal was reduced relative to the unmodified strain. These data are encouraging for the future testing and implementation of genetic control strategies and will help guide future field use of this and other engineered strains.

Field performance of engineered male mosquitoes.

Dengue is the most medically important arthropod-borne viral disease, with 50-100 million cases reported annually worldwide. As no licensed vaccine or dedicated therapy exists for dengue, the most promising strategies to control the disease involve targeting the predominant mosquito vector, Aedes aegypti. However, the current methods to do this are inadequate. Various approaches involving genetically engineered mosquitoes have been proposed, including the release of transgenic sterile males. However, the ability of laboratory-reared, engineered male mosquitoes to effectively compete with wild males in terms of finding and mating with wild females, which is critical to the success of these strategies, has remained untested. We report data from the first open-field trial involving a strain of engineered mosquito. We demonstrated that genetically modified male mosquitoes, released across 10 hectares for a 4-week period, mated successfully with wild females and fertilized their eggs. These findings suggest the feasibility of this technology to control dengue by suppressing field populations of A. aegypti.

Comparison of life history characteristics of the genetically modified OX513A line and a wild type strain of Aedes aegypti.

The idea of implementing genetics-based insect control strategies modelled on the traditional SIT (Sterile Insect Technique), such as RIDL (Release of Insects carrying a Dominant Lethal), is becoming increasingly popular. In this paper, we compare a genetically modified line of Aedes aegypti carrying a tetracycline repressible, lethal positive feedback system (OX513A) with a genetically similar, unmodified counterpart and their respective responses to increasing larval rearing density using a constant amount of food per larva. The parameters that we examined were larval mortality, developmental rate (i.e., time to pupation), adult size and longevity. Analysis revealed some statistically significant differences between the life history traits we examined. The genetically modified OX513A line overall showed 5% lower larval survival as well as reduced adult longevity (20 vs 24 days mean lifespan) compared to the unmodified line. Furthermore, the OX513A line pupated about one day sooner, which could be advantageous in mass-rearing, but produced somewhat smaller adults than the unmodified line; this effect was more pronounced in females than in males. Increasing the larval rearing density delayed pupation, decreased adult longevity and reduced adult size in both lines. While the delay in pupation and the decrease in longevity were similar between the two lines, the decrease in adult size was more pronounced for OX513A males.Our study shows that in a controlled laboratory situation the transgenic sterile OX513A line may have somewhat reduced performance compared to its unmodified counterpart and that high rearing densities may further reduce performance. Laboratory-based cage trials as well as field trials are necessary to assess how the differences in life history traits documented here impact the males' success upon release. Furthermore, this paper highlights the potential value of optimisation of mass-rearing systems as optimised rearing methods may be able to alleviate performance issues associated with specific lines or with lab-adapted lines in general.

Genetic elimination of dengue vector mosquitoes.

An approach based on mosquitoes carrying a conditional dominant lethal gene (release of insects carrying a dominant lethal, RIDL) is being developed to control the transmission of dengue viruses by vector population suppression. A transgenic strain, designated OX3604C, of the major dengue vector, Aedes aegypti, was engineered to have a repressible female-specific flightless phenotype. This strain circumvents the need for radiation-induced sterilization, allows genetic sexing resulting in male-only releases, and permits the release of eggs instead of adult mosquitoes. OX3604C males introduced weekly into large laboratory cages containing stable target mosquito populations at initial ratios of 8.5-101 OX3604Ctarget eliminated the populations within 10-20 weeks. These data support the further testing of this strain in contained or confined field trials to evaluate mating competitiveness and environmental and other effects. Successful completion of the field trials should facilitate incorporation of this approach into area-wide dengue control or elimination efforts as a component of an integrated vector management strategy.

Site-specific integration and expression of an anti-malarial gene in transgenic Anopheles gambiae significantly reduces Plasmodium infections.

Diseases transmitted by mosquitoes have a devastating impact on global health and this is worsening due to difficulties with existing control measures and climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. Historically the genetic modification of insects has relied upon transposable elements which have many limitations despite their successful use. To circumvent these limitations the Streptomyces phage phiC31 integrase system has been successfully adapted for site-specific transgene integration in insects. Here, we present the first site-specific transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 targeting site at a defined genomic location. A second phase of genetic modification then achieved site-specific integration of Vida3, a synthetic anti-malarial gene. Expression of Vida3, specifically in the midgut of bloodfed females, offered consistent and significant protection against Plasmodium yoelii nigeriensis, reducing average parasite intensity by 85%. Similar protection was observed against Plasmodium falciparum in some experiments, although protection was inconsistent. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters for their expression, enabling those offering maximum effect with minimum fitness cost to be identified. In the future, this technology will allow effective comparisons and informed choices to be made, potentially leading to complete transmission blockade.

Irritant and repellent behavioral responses of Aedes aegypti male populations developed for RIDL disease control strategies.

Behavioral responses of Aedes aegypti male populations developed for Release of Insects Carrying a Dominant Lethal (RIDL) technology and a Malaysian wild-type population of two age groups (4-5 and 8-10 d old) were tested under laboratory conditions against chemical irritants and repellents using the high-throughput screening system device. Results indicate that all male Ae. aegypti test populations showed significant (P < 0.01) behavioral escape responses when exposed to alphacypermethrin, DDT, and deltamethrin at the test dose of 25 nmol/cm2. In addition, all populations showed significant (P < 0.05) spatial repellent responses to DDT, whereas alphacypermethrin and deltamethrin elicited no directional movement in the assay. These data suggest that genetic modification has not suppressed expected irritancy and repellency behavior. Age effects were minimal in both contact irritant and spatial repellent assays. The magnitude of irritant response, based on percentage responding, was stronger in the RIDL test cohorts as compared with the wild-type Malaysian population, but the impact, if any, that this increased behavioral sensitivity might have on the success of a RIDL strategy has yet to be defined. Information of the type reported in the current study is vital in defining the effects of genetic modification on vector behavior and understanding how these behaviors may influence the success of RIDL technology as they relate to other vector control interventions implemented in the same disease-endemic locale.