Bacterial Larvicide, Bacillus thuringiensis israelensis Strain AM 65-52 Water Dispersible Granule Formulation Impacts Both Dengue Vector, Aedes aegypti (L.) Population Density and Disease Transmission in Cambodia.

A multi-phased study was conducted in Cambodia from 2005-2011 to measure the impact of larviciding with the bacterial larvicide, Bacillus thuringiensis israelensis (Bti), a water dispersible granule (WG) formulation on the vector, Aedes aegypti (L.) and the epidemiology. In our studies, all in-use containers were treated at 8 g/1000 L, including smaller containers and animal feeders which were found to contribute 23% of Ae aegypti pupae. The treated waters were subjected to routine water exchange activities. Pupal production was suppressed by an average 91% for 8 weeks. Pupal numbers continued to remain significantly lower than the untreated commune (UTC) for 13 weeks post treatment in the peak dengue vector season (p<0.05). Suppression of pupal production was supported by very low adult numbers in the treated commune. An average 70% of the household harbored 0-5 Ae aegypti mosquitoes per home for 8 weeks post treatment, but in the same period of time >50% of the household in the UTC harbored ≥11 mosquitoes per home. The adult population continued to remain at significantly much lower numbers in the Bti treated commune than in the UTC for 10-12 weeks post treatment (p<0.05). In 2011, a pilot operational program was evaluated in Kandal Province, a temephos resistant site. It was concluded that 2 cycles of Bti treatment in the 6 months monsoon season with complete coverage of the target districts achieved an overall dengue case reduction of 48% in the 6 treated districts compared to the previous year, 2010. Five untreated districts in the same province had an overwhelming increase of 352% of dengue cases during the same period of time. The larvicide efficacy, treatment of all in-use containers at the start of the monsoon season, together with treatment coverage of entire districts interrupted disease transmission in the temephos resistant province.

Spatial genetic structure of Aedes aegypti mosquitoes in mainland Southeast Asia.

Aedes aegypti mosquitoes originated in Africa and are thought to have spread recently to Southeast Asia, where they are the major vector of dengue. Thirteen microsatellite loci were used to determine the genetic population structure of A. aegypti at a hierarchy of spatial scales encompassing 36 sites in Myanmar, Cambodia and Thailand, and two sites in Sri Lanka and Nigeria. Low, but significant, genetic structuring was found at all spatial scales (from 5 to >2000 km) and significant F IS values indicated genetic structuring even within 500 m. Spatially dependent genetic-clustering methods revealed that although spatial distance plays a role in shaping larger-scale population structure, it is not the only factor. Genetic heterogeneity in major port cities and genetic similarity of distant locations connected by major roads, suggest that human transportation routes have resulted in passive long-distance migration of A. aegypti. The restricted dispersal on a small spatial scale will make localized control efforts and sterile insect technology effective for dengue control. Conversely, preventing the establishment of insecticide resistance genes or spreading refractory genes in a genetic modification strategy would be challenging. These effects on vector control will depend on the relative strength of the opposing effects of passive dispersal.

Pupal sampling for Aedes aegypti (L.) surveillance and potential stratification of dengue high-risk areas in Cambodia.

OBJECTIVES: To identify and describe the distribution of dengue vectors and factors affecting this distribution in Cambodia, with a view to practicing rational, evidence-based dengue outbreak prevention activities. METHODS: Entomological survey with a questionnaire component in 100 randomly selected households in each of 13 clusters of high or low human population density of seven Cambodian provinces. Entomological and other indices were calculated, and statistical methods used to describe factors of potential outbreak risk. RESULTS: Aedes aegypti was the principle dengue vector in all clusters, making up 95.5% (20,555 of 21,325) of the Aedes pupae population. The majority of pupae were recovered either from large concrete water storage jars (16,230; 76.1%) or concrete water storage tanks (2819; 13.2%). There were small but significantly higher levels of dengue vector infestation in rural than urban areas. The mean pupae density over the survey was 16.4/house, which ranged between clusters from 5.2/house to 56.9/house. The 'pupae-per-person' index was 2.4 and 3.6 in urban and rural areas, respectively, and was independent of mean human population density or household water container distribution. CONCLUSIONS: High populations of household-associated dengue vectors were present in all surveyed clusters. The highly skewed distribution of pupae in a limited number of key containers suggests adoption and further development of community-based control measures targeting these containers holds most potential chance of controlling dengue outbreaks in Cambodia.

Mitochondrial pseudogenes in the nuclear genome of Aedes aegypti mosquitoes: implications for past and future population genetic studies.

BACKGROUND: Mitochondrial DNA (mtDNA) is widely used in population genetic and phylogenetic studies in animals. However, such studies can generate misleading results if the species concerned contain nuclear copies of mtDNA (Numts) as these may amplify in addition to, or even instead of, the authentic target mtDNA. The aim of this study was to determine if Numts are present in Aedes aegypti mosquitoes, to characterise any Numts detected, and to assess the utility of using mtDNA for population genetics studies in this species. RESULTS: BLAST searches revealed large numbers of Numts in the Ae. aegypti nuclear genome on 146 supercontigs. Although the majority are short (80% < 300 bp), some Numts are almost full length mtDNA copies. These long Numts are not due to misassembly of the nuclear genome sequence as the Numt-nuclear genome junctions could be recovered by amplification and sequencing. Numt evolution appears to be a complex process in Ae. aegypti with ongoing genomic integration, fragmentation and mutation and the secondary movement of Numts within the nuclear genome.The PCR amplification of the putative mtDNA nicotinamide adenine dinucleotide dehydrogenase subunit 4 (ND4) gene from 166 Southeast Asian Ae. aegypti mosquitoes generated a network with two highly divergent lineages (clade 1 and clade 2). Approximately 15% of the ND4 sequences were a composite of those from each clade indicating Numt amplification in addition to, or instead of, mtDNA. Clade 1 was shown to be composed at least partially of Numts by the removal of clade 1-specific bases from composite sequences following enrichment of the mtDNA. It is possible that all the clade 1 sequences in the network were Numts since the clade 2 sequences correspond to the known mitochondrial genome sequence and since all the individuals that produced clade 1 sequences were also found to contain clade 2 mtDNA-like sequences using clade 2-specific primers. However, either or both sets of clade sequences could have Numts since the BLAST searches revealed two long Numts that match clade 2 and one long Numt that matches clade 1. The substantial numbers of mutations in cloned ND4 PCR products also suggest there are both recently-derived clade 1 and clade 2 Numt sequences. CONCLUSION: We conclude that Numts are prevalent in Ae. aegypti and that it is difficult to distinguish mtDNA sequences due to the presence of recently formed Numts. Given this, future population genetic or phylogenetic studies in Ae. aegypti should use nuclear, rather than mtDNA, markers.

Six months of Aedes aegypti control with a novel controlled-release formulation of pyriproxyfen in domestic water storage containers in Cambodia.

A field evaluation of a novel, 5% controlled-release formulation of pyriproxyfen was carried out in 400-500 liter concrete water storage jars, the most common and important larval habitat of the dengue vector, Aedes aegypti, in Cambodia. The formulation consisted of cylindrical resin strands, 3 mm in diameter and 40 mm in length. Pyriproxyfen was applied to 100 jars at a target dose of 0.03-0.04 mg of active ingredient (a.i.) per liter (30-40 ppb) in households in Phum Thmei, a village near the capital city of Phnom Penh, in April 2005. Inhibition of adult emergence (IE) in field populations ofAe. aegypti was measured every 2 weeks for 34 weeks. IE in treated jars exceeded 90% for 20 weeks, and remained above 80% until the end of the study. In 25 untreated jars, failure of pupae to metamorphose into viable adults remained below 4%. No alteration of taste or other undesirable effects of the treatment were reported by householders. This single treatment provided control of Ae. aegypti in water jars for the length of the main dengue transmission season in Cambodia that normally extends from May to November.

Community-based use of the larvivorous fish Poecilia reticulata to control the dengue vector Aedes aegypti in domestic water storage containers in rural Cambodia.

A community-based study of the distribution of larvivorous fish, Poecilia reticulata (common name: guppy), in water storage containers for dengue control was undertaken in 14 villages and approximately 1,000 households in Cambodia. Community volunteers reared guppies and distributed them in water jars and tanks in households for which they were responsible. A nearby control area received no intervention. One year after project commencement, 56.9% of eligible containers contained guppies and there was a 79.0% reduction in Aedes infestation in the intervention community compared with the control. Smaller or discarded containers unsuitable for guppy distribution in the intervention area also had 51% less infestation than those in the control area, suggesting a "community-wide" protective effect. In addition, there was less infestation in villages with higher rates of fish uptake, suggesting that the presence of fish was responsible for a reduction in Aedes infestation. This applied vector control model was well-accepted, effective, efficient, and shows promise as a sustainable community-based, non-insecticidal intervention for dengue vector control in large domestic water storage containers in rural Cambodia and elsewhere.

The effect of long-lasting insecticidal water container covers on field populations of Aedes aegypti (L.) mosquitoes in Cambodia.

Dengue in Cambodia is mainly transmitted by Aedes aegypti (L.) mosquitoes that primarily breed in large, concrete jars (> or =200 liters) used for the storage of water for domestic use. Following a preliminary risk assessment, long-lasting insecticidal netting (LN) treated with deltamethrin was incorporated into the design of the covers for these jars. Their effect on immature and adult female populations of Ae. aegypti in six villages in a peri-urban area of Cambodia were compared with populations in six nearby control villages before and for 22 weeks after distribution of the jar covers. There were significantly fewer pupae per house in intervention villages than in control villages (6.6 and 31.9, respectively, p<0.01). Fewer pupae were recovered from intervention houses than from control houses at every post-intervention assessment. Two weeks after the intervention, the average number of indoor resting female Ae. aegypti per house in the intervention villages had declined approximately three-fold, whereas in the controls there was only a slight reduction (16%). The magnitude of the difference between the two areas diminished over time, which contact bioassays confirmed was likely due to a gradual reduction of insecticidal effect of the jar covers. In the study area, insecticide-treated covers for large concrete water storage jars were efficacious for controlling Ae. aegypti in the protected water jars and with a demonstrable effect on adult densities and survival. Further studies of this targeted container strategy in Cambodia, and elsewhere, are recommended. However, improvements in technology that would extend the duration of insecticidal effectiveness of LN materials may be needed for the development of cost-effective public health applications.

Efficacy of Bacillus thuringiensis israelensis, VectoBac WG and DT, formulations against dengue mosquito vectors in cement potable water jars in Cambodia.

This study reports the evaluation of Bacillus thuringiensis israelensis (Bti), a biological larvicide, in cement jars holding river, well and rain water. Two Bti formulations, VectoBac WG and VectoBac DT, were evaluated in a village in Phnom Penh. Thirty-one households with cement jars supporting the colonization of Ae.aegypti immatures were chosen. In each house 3 jars were aligned next to each another and filled with the same type of water. One of the 3 jars was treated with VectoBac WG at 0.4 g per 50 liters, a second jar was treated with VectoBac DT at 1 tablet per 50 liters, and a third jar was an untreated control (UTC). The jars were not covered, kept outdoors and not subjected to water exchange activity. The efficacy of VectoBac to control natural Ae.aegypti infestation was measured by Ae.aegypti pupae surveillance, conducted 3 days per week for 3 months post-treatment (June-September 2004). All pupae were removed, allowed to emerge in the Cambodia National Malaria Center insectarium and the emerged adults were identified and counted. The VectoBac treatments were more effective in river water, followed by well and rain water. The VectoBac treatments significantly reduced the pupae numbers for a minimum of 3 months in the river water and 2.5 months in the well water (p < 0.05). In the rain water, the pupae densities in the VectoBac WG and DT treated jars were not significantly different from the untreated jars, although the treated jars yielded 2.0 to 5.2 fold less pupae, respectively, than in the untreated jars during the 3 months post-treatment. The efficacy of VectoBac WG to control Ae.aegypti was similar to the efficacy of VectoBac DT in the 3 water types (p > 0.05). It was also observed that VectoBac WG and DT were target specific, without any adverse effects on aquatic predatory insects common in well and rain water. VectoBac WG and DT were found to be easy-to-use formulations, with no need to repackage them prior to use in the containers. The amounts of VectoBac WG and DT used were 12.5 fold less by weight than temephos (Abate 1.0% SG).

Inhibition of adult emergence of Aedes aegypti in simulated domestic water-storage containers by using a controlled-release formulation of pyriproxyfen.

Concrete domestic water-storage jars are a common larval habitat of the dengue vector Aedes aegypti in countries of Southeast Asia. The efficacy of a novel controlled-release formulation of the insect growth regulator pyriproxyfen, designed to inhibit adult emergence for 6 months (the approximate duration of the main dengue transmission season in many endemic countries) was tested in Cambodia against a local strain of Ae. aegypti in 200-liter jars. The resin-based formulation contained 4.8% active ingredient (AI). At target dosages of 18, 27, and 36 ppb of AI, inhibition of adult emergence remained above 95% for at least 2 months. After 3 months at 18 ppb AI, the residual efficacy was significantly lower than for the higher dosages (P < 0.05). At the higher dosages, inhibition of adult emergence was > or = 87% for 6 months. At a dosage of 27 ppb AI, monthly removal and replacement of two thirds of the water did not reduce efficacy (P > 0.05). Potential operational advantages and challenges of using the formulation are discussed. Larger-scale efficacy studies are recommended in community settings.