A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium.

Wolbachia are maternally inherited intracellular bacterial symbionts that are estimated to infect more than 60% of all insect species. While Wolbachia is commonly found in many mosquitoes it is absent from the species that are considered to be of major importance for the transmission of human pathogens. The successful introduction of a life-shortening strain of Wolbachia into the dengue vector Aedes aegypti that halves adult lifespan has recently been reported. Here we show that this same Wolbachia infection also directly inhibits the ability of a range of pathogens to infect this mosquito species. The effect is Wolbachia strain specific and relates to Wolbachia priming of the mosquito innate immune system and potentially competition for limiting cellular resources required for pathogen replication. We suggest that this Wolbachia-mediated pathogen interference may work synergistically with the life-shortening strategy proposed previously to provide a powerful approach for the control of insect transmitted diseases.

Role of envelope N-linked glycosylation in Ross River virus virulence and transmission.

With an expanding geographical range and no specific treatments, human arthritogenic alphaviral disease poses a significant problem worldwide. Previous in vitro work with Ross River virus (RRV) demonstrated that alphaviral N-linked glycosylation contributes to type I IFN (IFN-αß) induction in myeloid dendritic cells. This study further evaluated the role of alphaviral N-linked glycans in vivo, assessing the effect of glycosylation on pathogenesis in a mouse model of RRV-induced disease and on viral infection and dissemination in a common mosquito vector, Aedes vigilax. A viral mutant lacking the E1-141 glycosylation site was attenuated for virus-induced disease, with reduced myositis and higher levels of IFN-γ induction at peak disease contributing to improved viral clearance, suggesting that glycosylation of the E1 glycoprotein plays a major role in the pathogenesis of RRV. Interestingly, RRV lacking E2-200 glycan had significantly reduced replication in the mosquito vector A. vigilax, whereas loss of either of the E1 or E2-262 glycans had little effect on the competence of the mosquito vector. Overall, these results indicate that glycosylation of the E1 and E2 glycoproteins of RRV provides important determinants of viral virulence and immunopathology in the mammalian host and replication in the mosquito vector.

Effect of Wolbachia on replication of West Nile virus in a mosquito cell line and adult mosquitoes.

Wolbachia as an endosymbiont is widespread in insects and other arthropods and is best known for reproductive manipulations of the host. Recently, it has been shown that wMelpop and wMel strains of Wolbachia inhibit the replication of several RNA viruses, including dengue virus, and other vector-borne pathogens (e.g., Plasmodium and filarial nematodes) in mosquitoes, providing an alternative approach to limit the transmission of vector-borne pathogens. In this study, we tested the effect of Wolbachia on the replication of West Nile Virus (WNV). Surprisingly, accumulation of the genomic RNA of WNV for all three strains of WNV tested (New York 99, Kunjin, and New South Wales) was enhanced in Wolbachia-infected Aedes aegypti cells (Aag2). However, the amount of secreted virus was significantly reduced in the presence of Wolbachia. Intrathoracic injections showed that replication of WNV in A. aegypti mosquitoes infected with wMel strain of Wolbachia was not inhibited, whereas wMelPop strain of Wolbachia significantly reduced the replication of WNV in mosquitoes. Further, when wMelPop mosquitoes were orally fed with WNV, virus infection, transmission, and dissemination rates were very low in Wolbachia-free mosquitoes and were completely inhibited in the presence of Wolbachia. The results suggest that (i) despite the enhancement of viral genomic RNA replication in the Wolbachia-infected cell line the production of secreted virus was significantly inhibited, (ii) the antiviral effect in intrathoracically infected mosquitoes depends on the strain of Wolbachia, and (iii) replication of the virus in orally fed mosquitoes was completely inhibited in wMelPop strain of Wolbachia.

Efficacy of residual insecticide Biflex AquaMax applied as barrier treatments for managing mosquito populations in suburban residential properties in southeast Queensland.

The pyrethroid insecticide Biflex AquaMax (bifenthrin) was evaluated for its ability to suppress coastal mosquito populations in a controlled suburban backyard study in Mango Hill, Queensland, Australia. The insecticide was applied to perimeter vegetation, fencing in selected backyards, or both, and mosquito populations were monitored weekly from 3 wk before to 8 wk after treatment (11 wk total) by using CDC miniature light traps and human bait landing rates. In addition, bioassays were conducted on vegetation and wood surfaces to monitor residual activity. The two most common species in light trap collections were the Ross River Virus vectors Aedes vigilax (Skuse) and Culex annulirostris Skuse. After treatment, Ae. vigilax populations in treated properties were significantly lower (75-90%) compared with untreated properties. In contrast, Cx. annulirotris, Coquillettidia xanthogaster (Edwards) and Mansonia uniformis (Theobald) populations were not impacted by the treatment. Bioassays revealed that the product gave better residual mortality when applied to vegetation (94.6 +/- 11.8%) than wooden surfaces (75.9 +/- 29.1%) during the 8-wk posttreatment period.

Impacts of Wolbachia infection on predator prey relationships: evaluating survival and horizontal transfer between wMelPop infected Aedes aegypti and its predators.

The wMelPop strain of Wolbachia is currently being investigated for its potential use as a biological control agent to reduce the ability of Aedes aegypti (L.) mosquitoes to transmit dengue viruses. The survival of a potential wMelPop infected Ae. aegypti strain for field release is important as a higher susceptibility to predation in the wMelPop strain could result in difficulties in achieving fixation. We investigated immature and adult survival as a function of susceptibility to predation by six naturally occurring predator species; cyclopoid copepods, fish, predatory Toxorhynchites mosquito larvae and a salticid jumping spider. The trials indicated that wMelPop infected and uninfected Ae. aegypti larvae and adults were equally susceptible to predation to all six tested predators. In addition to evaluating any potential fitness costs to the infected host, we were unable to demonstrate horizontal transfer of wMelPop via consumption of infected Ae. aegypti larvae to the above predators. That susceptibility to predation was consistent across mosquito life stage, predator species and experimental venue is strong evidence that despite the neurotrophic and extensive nature of wMelPop infection, behavioral changes are not occurring, or at least not a determining factor in survival when exposed to a predator. Based on our results and the ecology of Wolbachia and mosquito predators, horizontal transfer of wMelPop from Ae. aegypti into naturally occurring predators is not cause for concern.

Investigation of environmental influences on a transcriptional assay for the prediction of age of Aedes aegypti (Diptera: Culicidae) mosquitoes.

We examined the effects of environmental regulation of gene transcription on the accuracy of a transcriptional profiling method for determining insect age. In combined temperature/nutrition treatments, Aedes aegypti (L.) mosquitoes were maintained in the laboratory at three different temperatures (20, 26, and 32 degrees C), and larvae were fed on low, medium, and high diet regimens. Adult mosquitoes of distinct size classes were produced. Transcription of three age-responsive genes (Ae-15848, Ae-8505, and Ae-4274) was measured from 1-, 10-, and 19-d-old specimens using a quantitative reverse-transcription polymerase chain reaction method incorporating dual-labeled TaqMan probes. Temperature had a significant effect on transcript abundance for two of the model genes (Ae-15848 and Ae-8505), and transcription of model genes was unaffected by the main effect of larval diet level; however, significant temperature by diet level interactions were observed. Total RNA yield from individual mosquitoes varied according to adult age and temperature, and when combined with wing length, provided a useful predictor variable in age prediction models. More accurate age predictions were achieved from models generated at the same temperature as test mosquitoes; however, whereas significant differences in mean predicted ages were observed between 1- and 10-d-old mosquitoes, differences between 10 and 19 d were nonsignificant. This study highlights the effect of environmental regulation on gene transcription age grading and the need to identify additional gene biomarkers of age to improve the classification of older mosquitoes.

Quantitative relationships between immature and emergent adult Aedes aegypti (Diptera: Culicidae) populations in water storage container habitats.

Although quantitative surveillance data for immature stages of Aedes aegypti are often used to prioritize containers or specific types of containers for control, the relationship between immature and emergent adult populations under field conditions is largely unknown. We examined the relationships between abundance of III/IV instars and pupae, and emerging adult population for a series of water storage containers in southern Vietnam. A large proportion of III/IV instars failed to progress to adulthood, and the relationships between III/IV instars and adults were poor. Collected IV instars appeared to be nutritionally deprived, although their size and nutrient levels were not reliable indicators of emergence success. Conversely, pupal abundance was a good indicator of emerging adult populations, especially over the ensuing 48-h period. Although there were clear advantages of pupal surveillance over surveillance of III/IV instars for the estimation of adult mosquito productivity, there were practical limitations associated with the enumeration of pupae, and their comparatively low densities may preclude the identification of potentially productive containers.

Quality assurance of aerial applications of larvicides for mosquito control: effects of granule and catch tray size on field monitoring programs.

Aerial applications of granular insecticides are preferable because they can effectively penetrate vegetation, there is less drift, and no loss of product due to evaporation. We aimed to 1) assess the field efficacy ofVectoBac G to control Aedes vigilax (Skuse) in saltmarsh pools, 2) develop a stochastic-modeling procedure to monitor application quality, and 3) assess the distribution of VectoBac G after an aerial application. Because ground-based studies with Ae. vigilax immatures found that VectoBac G provided effective control below the recommended label rate of 7 kg/ha, we trialed a nominated aerial rate of 5 kg/ha as a case study. Our distribution pattern modeling method indicated that the variability in the number of VectoBac G particles captured in catch-trays was greater than expected for 5 kg/ha and that the widely accepted contour mapping approach to visualize the deposition pattern provided spurious results and therefore was not statistically appropriate. Based on the results of distribution pattern modeling, we calculated the catch tray size required to analyze the distribution of aerially applied granular formulations. The minimum catch tray size for products with large granules was 4 m2 for Altosid pellets and 2 m2 for VectoBac G. In contrast, the minimum catch-tray size for Altosid XRG, Aquabac G, and Altosand, with smaller granule sizes, was 1 m2. Little gain in precision would be made by increasing the catch-tray size further, when the increased workload and infrastructure is considered. Our improved methods for monitoring the distribution pattern of aerially applied granular insecticides can be adapted for use by both public health and agricultural contractors.

Habitat characteristics and eggshell distribution of the salt marsh mosquito, Aedes vigilax, in marshes in subtropical Eastern Australia.

Research at 10 locations in coastal subtropical Queensland, Australia, has shown that salt marshes contained heterogeneous distributions of eggshells of the pest and vector mosquito Aedes vigilax (Skuse) (Diptera:Culicidae). The eggshell distribution was related to specific vegetation assemblages, with a mix of the grass, Sporobolus virginicus (L.) Kunth (Poales: Poaceae), and the beaded glasswort, Sarcocornia quinqueflora (Bunge ex (Ung.-Stern) A.J. Scott (Caryophyllales: Chenopodiaceae), as significantly higher in eggshells than any other vegetation. There were also high numbers in the mix of S. virginicus with the arrowgrass, Triglochin striata Ruiz & Pavón (Alismatales: Juncaginaceae). Both mixed types are found in relatively wetter areas, despite very few eggshells being found generally in the low marsh. Most sites contained S. virginicus and eggshell locations were variable for this species alone. This was probably related to its life form variability in response to salinity and location on the marsh. Location on the marsh was important for eggshell distribution with most eggshells around the edges of pools and depressions, followed by, but to a significantly lesser extent, the marsh surface. Eggshells were fewest in the low marsh. Partition analysis resulted in a tree that simplified and summarised the factors important for eggshell distribution confirming the individual analyses. The potential effects of climate, sea level and other change are also briefly discussed in the context of likely changes to land cover and relative location on the marsh. For example, increased sea level may lead to low marsh conditions extending into higher marsh area with implications for oviposition and numbers of eggshells.

Mosquito feeding patterns and natural infection of vertebrates with Ross River and Barmah Forest viruses in Brisbane, Australia.

Host feeding patterns of mosquitoes were assessed through the identification of 865 blood meals collected from Brisbane during 2000-2001. Under natural conditions, mosquito feeding (including that of Culex annulirostris, Aedes vigilax, and Aedes notoscriptus) was primarily on dogs (37.4%), but also on birds (18.4%), horses (16.8%), brushtail possums (13.3%), humans (11.6%), and cats, flying foxes, and macropods, depending on site. From 1997 to 1999, sera (N=1706) were collected from dogs, cats, horses, flying foxes, and brushtail possums in the Brisbane area and were analyzed by microneutralization assay for antibodies to Ross River virus (RRV) and Barmah Forest virus (BFV). For RRV, all vertebrate species tested had been naturally infected, and seroprevalence varied from 10.5% to 25.5%, whereas for BFV, rates varied between 0% and 11.3%. Brushtail possums were often infected in the field, with 17.6% and 10.7% of wild individuals having antibodies to RRV and BFV, respectively. Horses and flying foxes also had a relatively high prevalence of antibodies to RRV. This study, therefore, provides data to indicate that brushtail possums play a role in the urban transmission of RRV in Brisbane and that horses, when they occur, also fill the same role.

Critical evaluation of quantitative sampling methods for Aedes aegypti (Diptera: Culicidae) immatures in water storage containers in Vietnam.

In response to an identified paucity of information on the size and composition of immature Aedes aegypti (L.) (Diptera: Culicidae) populations in large field containers, we assessed net sampling and pumping/sieving methods for estimating and enumerating third (III)/fourth (IV) instar and pupal populations. Sweep net detection thresholds (number above which > or = 90% chance of a positive sample) were < or = 28 immatures for seven different container types (115-3000 liter jars and tanks) in the laboratory, and mean recovery percentages varied by container type (6.15-41.29 and 7.39-33.10% for III/IV instars and pupae, respectively). A pumping method or hand bailing was applied in the field for the collection of III/IV instars and pupae from 406 receptacles, of which 343 had been previously sampled via a five-sweep netting technique. Larvae were 9.30 times more prevalent than pupae, and abundance varied by container type with means of 36-537 III/IV instars and 6-53 pupae per receptacle. Sweep netting for III/IV instars effectively identified 86.2% of Ae. aegypti-positive containers, whereas sampling for pupae detected only 43.1% of positive containers. When conversion factors (inverse of laboratory recovery percentages) were applied to field net sampling data, estimates of container populations were more accurate for III/IV instars than pupae (maximum R2 = 0.610 and 0.328, respectively); however, the relationship between immature abundance and emergent adult populations remains to be defined.

Control of arbovirus vector Verrallina funerea (Diptera: Culicidae) in southeast Queensland, Australia.

In Australia, the brackish water mosquito Verrallina funerea (Theobald) (Diptera: Culicidae) is a serious pest and vector of Ross River and Barmah Forest viruses in coastal areas in the northeast of the continent. We report on the first extensive evaluation of the efficacy of various pesticides against this species, including laboratory dose-response assays and small plot field trials. In the laboratory, Ve. funerea was susceptible to temephos (Abate 100E) (lethal concentration, 95% endpoint [LC95] of 1.651 ppb), Bacillus thuringiensis subsp. israelensis de Barjac (Bti) (VectoBac 12AS) (LC95 of 0.033 ppm), s-methoprene (ProLink Liquid Larvicide [PLL]) (emergence inhibition, 95% endpoint [EI95] of 2.647 ppb), and technical grade pyriproxyfen (EI95 of 0.227 ppb). In contrast, Ve.funerea was highly refractory to Bacillus sphaericus Neide (VectoLex WG) (LC95 of >855.3 ppm). The survival of adults exposed as larvae to sublethal doses of s-methoprene was reduced for those doses greater than the EI50, although sex ratios were unaffected. Field trials demonstrated that Ve. funerea was susceptible to Bti (VectoBac G at 7 kg/ha) and s-methoprene (ProLink ProSand at 4 kg/ha), with these products giving >99 and >98% control, respectively. We also observed a significant increase in immature mortality in s-methoprene-treated pools, suggesting that s-methoprene field trials based solely on emergence inhibition of pupae may underestimate the efficacy of this insecticide.

Investigation of cuticular hydrocarbons for determining the age and survivorship of australasian mosquitoes.

The cuticular hydrocarbon (CH) technique of age grading mosquitoes was evaluated for use on laboratory-reared Aedes aegypti (L.), Anopheles farauti (Laveran), and Ochlerotatus vigilax (Skuse). Gas chromatography/mass spectroscopy was used to determine the relative abundance of five alkanes in hexane extracts from the legs of individual mosquitoes. Age-related changes to relative hydrocarbon abundances were observed from An. farauti and Ae. aegypti, which are vectors of malaria and dengue, respectively. Female An. farauti were classified into two age categories (1-5 and >/= 5 days old at 27 degrees C) and Ae. aegypti into three age categories (1 to < 5, 5 to < 9 and >/= 9 days old at 27 degrees C) based on these changes. However, there was an absence of predicable age-related changes to hydrocarbon abundance in Oc. vigilax. Simulation modeling was used to construct sequential sampling guidelines for the application of this technique to estimate the survivorship of Ae. aegypti and An. farauti populations. These guidelines define the relationship between the survival rate, number of mosquitoes sampled, CH-based predictions of age, and the accuracy of survival rate estimates. They demonstrated, for example, that if 19% of a population of Ae. aegypti is estimated to be >/= 9 days old by CH analysis, an estimate of the daily survival rate from the exponential model should be based on a sample of 200 mosquitoes for the survival rate estimate to be within 5% of the actual rate. However, if only 10% of the population is estimated to be >/= 9 days old, 500 mosquitoes would need to be analyzed for the survival rate estimate to be of equivalent accuracy.

Role of Verrallina funerea (Diptera: Culicidae) in transmission of Barmah Forest virus and Ross River virus in coastal areas of eastern Australia.

Verrallina funerea (Theobald) (Diptera: Culicidae) is a brackish water mosquito species found most commonly in Indonesia, Papua New Guinea, and the northeastern coastal regions of Australia. Aspects of the vector competence of this species for Barmah Forest virus (family Togaviridae, genus Alphavirus, BFV) and Ross River virus (family Togaviridae, genus Alphavirus, RRV), two medically important arboviruses in Australia, were investigated. Laboratory-reared Ve. funerea were moderately susceptible to experimental infection with BFV (median cell culture infectious dose of 10(3.6) per mosquito) and were capable of transmission to suckling mice (52% after a 9-12-d extrinsic incubation period). Maximum salivary gland infections for BFV (65%) and RRV (50%) were observed 8 and 10 d postinfection, respectively. To examine any regional differences in vector competence, field populations (separated by up to 200 km) of Ve. funerea and Aedes vigilax (Skuse) from southeastern Queensland and northern New South Wales were fed BFV and RRV, and subsequent infection rates were compared. For both viruses, no statistically significant variations in body, disseminated, or salivary gland infection rates were found in either Ve. funerea or Ae. vigilax. The results from this study indicate that Ve. funerea may have an important role as an amplification vector during outbreaks of both viruses and that local government authorities should rapidly treat brackish water habitats to control this species during periods of increased disease activity.

Laboratory evaluation of the effect of alternative prey and vegetation on predation of Culex annulirostris immatures by Australian native fish species.

The impact of alternative prey and simulated vegetation on Culex annulirostris Skuse predation efficacy by Australian smelt, Retropinna semoni (Retropinnidae); crimson-spotted rainbowfish, Melanotaenia duboulayi (Melanotaeniidae); empire gudgeon, Hypseleotris compressa (Eleotridae); estuary perchlet, Ambassis marianus (Ambassidae); firetail gudgeon, Hypseleotris galii (Eleotridae); fly-specked hardyhead, Craterocephalus stercusmuscarum (Atherinidae); and Pacific blue-eye, Pseudomugil signifer (Atherinidae), was evaluated in Queensland, Australia. The presence of chironomid midge larvae and tusked frog, Adelotus brevis (Leptodactylidae), tadpoles did not have a significant negative impact on the predation rates of Cx. annulirostris by these 7 fish species. Hypseleotris galii, M. duboulayi, and R. semoni demonstrated strong preference for larvae of Cx. annulirostris over both alternative prey species. In the presence of alternative prey species, the mean predation rate of M. duboulayi on larvae of Cx. annulirostris remained greater than that of other fish species tested. When evaluated at varying densities of simulated vegetation, predation rates of all fish species were similar to those reported in open conditions.

Evaluation of Melanotaenia duboulayi (Atheriniformes: Melanotaeniidae), Hypseleotris galli (Perciformes: Eleotridae), and larvicide VectoLex WG (Bacillus sphaericus) for integrated control of Culex annulirostris.

Australian freshwater fish species Melanotaenia duboulayi and Hypseleotris galii were selected for a small plot field evaluation of an integrated pest management strategy using native fish and VectoLex WG (Bacillus sphaericus) for the control of Culex annulirostris Skuse, the principal freshwater vector of arbovirus Ross River virus in Australia. When tested alone, the level of control afforded by M. duboulayi and H. galii was highly dependent on the prerelease density of mosquito larvae; and even when stocking rates as high as 10 g per pond (>30 kg/ha) were used, larval abundance was too high to attain adequate control from fish alone. In contrast, treatment with VectoLex WG at 500 g/ha resulted in 100% mortality of Cx. annulirostris immatures, but no residual activity was evident. The delayed reduction of Cx. annulirostris immatures in ponds stocked with fish alone, and the recolonization by Cx. annulirostris in ponds after treatment with B. sphaericus, did not occur when both treatments were combined.

Effects of Larval Nutrition on Wolbachia-Based Dengue Virus Interference in Aedes aegypti (Diptera: Culicidae).

In order to assess the broad-scale applicability of field releases of Wolbachia for the biological control of insect-transmitted diseases, we determined the relationship between the larval diet of Aedes aegypti L. mosquitoes infected with Wolbachia strains and their susceptibility to dengue virus (DENV) infection via intrathoracic injection and oral inoculation. Larvae were reared on diets that varied in the quantity of food which had the effect of modifying development time and adult body size. Wolbachia wMel infection was associated with highly significant reductions in dengue serotype 2 (DENV-2) infection rates of between 80 and 97.5% following intrathoracic injection of adults emerging from three diet levels. Reductions were 100% in two diet level treatments following oral inoculation. Similarly, wMelPop infection was associated with highly significant reductions in DENV-2 infection rates of between 95 and 100% for intrathoracic injection and 97.5 and 100% for oral inoculation across diet level treatments. Larval diet level had no significant effect on DENV-2 infection rates in the presence of Wolbachia infection in mosquitoes that were intrathoracically injected with the virus. This indicates that the effectiveness of Wolbachia on vector competence disruption within Ae. aegypti is unlikely to be compromised by variable larval nutrition in field settings.

Environmental predictors of Ross River virus disease outbreaks in Queensland, Australia.

Ross River virus (RRV) disease is the most common mosquito-borne disease in Australia, with the majority of cases reported from Queensland. In this study we investigate the relationship between local RRV disease outbreaks and standardized rainfall and temperature data in Queensland. No one set of variables could be found to accurately predict RRV disease outbreaks across all of Queensland, although good predictive models could be developed for smaller regions. The variables identified as important in predicting RRV disease outbreaks differed between regions, and also between summer and autumn. This work highlights the sensitive relationship between virus prevalence, mosquito bionomics, and climate, illustrating that critical climatic factors differ depending on underlying environmental conditions. Identification of factors leading to RRV disease outbreaks will help local authorities identify periods of high risk, optimizing the provision of additional mosquito control measures.

Elimination of dengue by community programs using Mesocyclops(Copepoda) against Aedes aegypti in central Vietnam.

From September 2000 to June 2003, a community-based program for dengue control using local predacious copepods of the genus Mesocyclops was conducted in three rural communes in the central Vietnam provinces of Quang Nam, Quang Ngai, and Khanh Hoa. Post-project, three subsequent entomologic surveys were conducted until March 2004. The number of households and residents in the communes were 5,913 and 27,167, respectively, and dengue notification rates for these communes from 1996 were as high as 2,418.5 per 100,000 persons. Following knowledge, attitude, and practice evaluations, surveys of water storage containers indicated that Mesocyclops spp. already occurred in 3-17% and that large tanks up to 2,000 liters, 130-300-liter jars, wells, and some 220-liter metal drums were the most productive habitats for Aedes aegypti. With technical support, the programs were driven by communal management committees, health collaborators, schoolteachers, and pupils. From quantitative estimates of the standing crop of third and fourth instars from 100 households, Ae. aegypti were reduced by approximately 90% by year 1, 92.3-98.6% by year 2, and Ae. aegypti immature forms had been eliminated from two of three communes by June 2003. Similarly, from resting adult collections from 100 households, densities were reduced to 0-1 per commune. By March 2004, two communes with no larvae had small numbers but the third was negative; one adult was collected in each of two communes while one became negative. Absolute estimates of third and fourth instars at the three intervention communes and one left untreated had significant correlations (P = 0.009-< 0.001) with numbers of adults aspirated from inside houses on each of 15 survey periods. By year 1, the incidence of dengue disease in the treated communes was reduced by 76.7% compared with non-intervention communes within the same districts, and no dengue was evident in 2002 and 2003, compared with 112.8 and 14.4 cases per 100,000 at district level. Since we had similar success in northern Vietnam from 1998 to 2000, this study demonstrates that this control model is broadly acceptable and achievable at community level but vigilance is required post-project to prevent reinfestation.

Mass spectrometry identification of age-associated proteins from the malaria mosquitoes Anopheles gambiae s.s. and Anopheles stephensi.

This study investigated proteomic changes occurring in Anopheles gambiae and Anopheles stephensi during adult mosquito aging. These changes were evaluated using two-dimensional difference gel electrophoresis (2D-DIGE) and the identities of aging related proteins were determined using capillary high-pressure liquid chromatography (capHPLC) coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometry (MS). Here, we have described the techniques used to determine age associated proteomic changes occurring in heads and thoraces across three age groups; 1, 9 and 17 d old A. gambiae and 4 age groups; 1, 9, 17 and 34 d old A. stephensi. We have provided normalised spot volume raw data for all protein spots that were visible on 2D-DIGE images for both species and processed Orbitrap mass spectrometry data. For public access, mass spectrometry raw data are available via ProteomeXchange with identifier PXD002153. A detailed description of this study has been described elsewhere [1].