A new robust diagnostic polymerase chain reaction for determining the mating status of female Anopheles gambiae mosquitoes.

The principal malaria vector in Africa, Anopheles gambiae, contains two pairs of autosomes and one pair of sex chromosomes. The Y chromosome is only associated with males and other Y chromosome-specific DNA sequences, which are transferred to women during mating. A reliable tool to determine the mating status of dried wild An. gambiae females is currently lacking. DNA was extracted from dried virgin and mated females and used to test whether Y chromosome-specific polymerase chain reaction (PCR) markers can be successfully amplified and used as a predictor of mating. Here we report a new PCR-based method to determine the mating status among successfully inseminated and virgin wild An. gambiae females, using three male-specific primers. This dissection-free method has the potential to facilitate studies of both population demographics and gene flow from dried mosquito samples routinely collected in epidemiologic monitoring and aid existing and new malaria-vector control approaches

Entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana reduce the survival of Xenopsylla brasiliensis larvae (Siphonaptera: Pulicidae).

Entomopathogenic fungi, particularly those belonging to the genera Metarhizium and Beauveria have shown great promise as arthropod vector control tools. These agents, however, have not been evaluated against flea vectors of plague. A 3-h exposure to the fungi coated paper at a concentration of 2 × 108 conidia m-2 infected >90% of flea larvae cadavers in the treatment groups. The infection reduced the survival of larvae that had been exposed to fungus relative to controls. The daily risk of dying was four- and over three-fold greater in larvae exposed to M. anisopliae (HR = 4, p<0.001) and B. bassiana (HR = 3.5, p<0.001) respectively. Both fungi can successfully infect and kill larvae of X. brasiliensis with a pooled median survival time (MST±SE) of 2±0.31 days post-exposure. These findings justify further research to investigate the bio-control potential of entomopathogenic fungi against fleas.\ud \u

Mesocosm experiments reveal the impact of mosquito control measures on malaria vector life history and population dynamics

The impact of control measures on mosquito vector fitness and demography is usually estimated from bioassays or indirect variables in the field. Whilst indicative, neither approach is sufficient to quantify the potentially complex response of mosquito populations to combined interventions. Here, large replicated mesocosms were used to measure the population-level response of the malaria vector Anopheles arabiensis to long-lasting insecticidal nets (LLINs) when used in isolation, or combined with insecticidal eave louvers (EL), or treatment of cattle with the endectocide Ivermectin (IM). State-space models (SSM) were fit to these experimental data, revealing that LLIN introduction reduced adult mosquito survival by 91% but allowed population persistence. ELs provided no additional benefit, but IM reduced mosquito fecundity by 59% and nearly eliminated all populations when combined with LLINs. This highlights the value of IM for integrated vector control, and mesocosm population experiments combined with SSM for identifying optimal combinations for vector population elimination

Establishment of a self-propagating population of the African malaria vector Anopheles arabiensis under semi-field conditions

Background: The successful control of insect disease vectors relies on a thorough understanding of their ecology and behaviour. However, knowledge of the ecology of many human disease vectors lags behind that of agricultural pests. This is partially due to the paucity of experimental tools for investigating their ecology under natural conditions without risk of exposure to disease. Assessment of vector life-history and demographic traits under natural conditions has also been hindered by the inherent difficulty of sampling these seasonally and temporally varying populations with the limited range of currently available tools. Consequently much of our knowledge of vector biology comes from studies of laboratory colonies, which may not accurately represent the genetic and behavioural diversity of natural populations. Contained semi-field systems (SFS) have been proposed as more appropriate tools for the study of vector ecology. SFS are relatively large, netting-enclosed, mesocosms in which vectors can fly freely, feed on natural plant and vertebrate host sources, and access realistic resting and oviposition sites. Methods: A self-replicating population of the malaria vector Anopheles arabiensis was established within a large field cage (21 x 9.1 x 7.1 m) at the Ifakara Health Institute, Tanzania that mimics the natural habitat features of the rural village environments where these vectors naturally occur. Offspring from wild females were used to establish this population whose life-history, behaviour and demography under semi-field conditions was monitored over 24 generations. Results: This study reports the first successful establishment and maintenance of an African malaria vector population under SFS conditions for multiple generations (&gt; 24). The host-seeking behaviour, time from blood feeding to oviposition, larval development, adult resting and swarming behaviour exhibited by An. arabiensis under SFS conditions were similar to those seen in nature. Conclusions: This study presents proof-of-principle that populations of important African malaria vectors can be established within environmentally realistic, contained semi-field settings. Such SFS will be valuable tools for the experimental study of vector ecology and assessment of their short-term ecological and longer-term evolutionary responses to existing and new vector control interventions

Clarification of anomalies in the application of a 2La molecular karyotyping method for the malaria vector Anopheles gambiae

BACKGROUND:Chromosomal inversions have been considered to be potentially important barriers to gene flow in many groups of animals through their effect on recombination suppression in heterokaryotypic individuals. Inversions can also enhance local adaptation in different groups of organisms and may often represent species-specific differences among closely related taxa. We conducted a study to characterize the 2La inversion karyotypes of An. gambiae sensu stricto mosquitoes sampled from the Kilombero Valley (Tanzania) using a newly designed PCR assay.RESULTS:We frequently encountered a (687 bp) fragment which was only present in the Kilombero Valley populations. Laboratory crossing between An. gambiae s.s. from Njage (Tanzania) and Kisumu (Western Kenya) populations resulted in F1 offspring carrying the observed fragment. Karyotype analysis did not indicate differences in 2La region chromosome morphology between individuals carrying the PCR fragments, the 207 bp fragment, or the 687 bp fragement.CONCLUSION:The observed insertion/deletion polymorphism within the region amplified by the 2La PCR diagnostic test may confound the interpretation of this assay and should be well considered in order to maintain an acceptable level of reliability in studies using this assay to describe the distribution and frequency of the 2La inversion among natural populations of An. gambiae s.

Does Cattle Milieu Provide a Potential Point to Target Wild Exophilic Anopheles arabiensis

Background. Anopheles arabiensis is increasingly dominating malaria transmission in Africa. The exophagy in mosquitoes threatens the effectiveness of indoor vector control strategies. This study aimed to evaluate the effectiveness of fungus against An. arabiensis when applied on cattle and their environments. Methods. Experiments were conducted under semi-field and small-scale field conditions within Kilombero valley. The semi-field reared females of 5–7 days old An. arabiensis were exposed to fungus-treated and untreated calf. Further, wild An. arabiensis were exposed to fungus-treated calves, mud-huts, and their controls. Mosquitoes were recaptured the next morning and proportion fed, infected, and survived were evaluated. Experiments were replicated three times using different individuals of calves. Results. A high proportion of An. arabiensis was fed on calves (>0.90) and become infected (0.94) while resting on fungus-treated mud walls than on other surfaces. However, fungus treatments reduced fecundity and survival of mosquitoes. Conclusion. This study demonstrates for the first time the potential of cattle and their milieu for controlling An. arabiensis. Most of An. arabiensis were fed and infected while resting on fungus-treated mud walls than on other surfaces. Fungus treatments reduced fecundity and survival of mosquitoes. These results suggest deployment of bioinsecticide zooprophylaxis against exophilic An. arabiensis

Eave tubes for malaria control in Africa : Initial development and semi-field evaluations in Tanzania Lucy Tusting, Jo Lines

Background: Presented here are a series of preliminary experiments evaluating "eave tubes" - a technology that combines house screening with a novel method of delivering insecticides for control of malaria mosquitoes. Methods: Eave tubes were first evaluated with overnight release and recapture of mosquitoes in a screened compartment containing a hut and human sleeper. Recapture numbers were used as a proxy for overnight survival. These trials tested physical characteristics of the eave tubes (height, diameter, angle), and different active ingredients (bendiocarb, LLIN material, fungus). Eave tubes in a hut with closed eaves were also compared to an LLIN protecting a sleeper in a hut with open eaves. Eave tubes were then evaluated in a larger compartment containing a self-replicating mosquito population, vegetation, and multiple houses and cattle sheds. In this "model village", LLINs were introduced first, followed by eave tubes and associated house modifications. Results: Initial testing suggested that tubes placed horizontally and at eave height had the biggest impact on mosquito recapture relative to respective controls. Comparison of active ingredients suggested roughly equivalent effects from bendiocarb, LLIN material, and fungal spores (although speed of kill was slower for fungus). The impact of treated netting on recapture rates ranged from 50 to 70 % reduction relative to controls. In subsequent experiments comparing bendiocarb-treated netting in eave tubes against a standard LLIN, the effect size was smaller but the eave tubes with closed eaves performed at least as well as the LLIN with open eaves. In the model village, introducing LLINs led to an approximate 60 % reduction in larval densities and 85 % reduction in indoor catches of host-seeking mosquitoes relative to pre-intervention values. Installing eave tubes and screening further reduced larval density (93 % relative to pre intervention values) and virtually eliminated indoor host-seeking mosquitoes. When the eave tubes and screening were removed, larval and adult catches recovered to pre-eave tube levels. Conclusions: These trials suggest that the "eave tube" package can impact overnight survival of host-seeking mosquitoes and can suppress mosquito populations, even in a complex environment. Further testing is now required to evaluate the robustness of these findings and demonstrate impact under field conditions

Colonization of malaria vectors under semi-field conditions as a strategy for maintaining genetic and phenotypic similarity with wild populations.

BackgroundMalaria still accounts for an estimated 207 million cases and 627,000 deaths worldwide each year. One proposed approach to complement existing malaria control methods is the release of genetically-modified (GM) and/or sterile male mosquitoes. As opposed to laboratory colonization, this requires realistic semi field systems to produce males that can compete for females in nature. This study investigated whether the establishment of a colony of the vector Anopheles arabiensis under more natural semi-field conditions can maintain higher levels of genetic diversity than achieved by laboratory colonization using traditional methods.MethodsWild females of the African malaria vector An. arabiensis were collected from a village in southern Tanzania and used to establish new colonies under different conditions at the Ifakara Health Institute. Levels of genetic diversity and inbreeding were monitored in colonies of An. arabiensis that were simultaneously established in small cage colonies in the SFS and in a large semi-field (SFS) cage and compared with that observed in the original founder population. Phenotypic traits that determine their fitness (body size and energetic reserves) were measured at 10(th) generation and compared to founder wild population.ResultsIn contrast to small cage colonies, the SFS population of An. arabiensis exhibited a higher degree of similarity to the founding field population through time in several ways: (i) the SFS colony maintained a significantly higher level of genetic variation than small cage colonies, (ii) the SFS colony had a lower degree of inbreeding than small cage colonies, and (iii) the mean and range of mosquito body size in the SFS colony was closer to that of the founding wild population than that of small cage colonies. Small cage colonies had significantly lower lipids and higher glycogen abundances than SFS and wild population.ConclusionsColonization of An. arabiensis under semi-field conditions was associated with the retention of a higher degree of genetic diversity, reduced inbreeding and greater phenotypic similarity to the founding wild population than observed in small cage colonies. Thus, mosquitoes from such semi-field populations are expected to provide more realistic representation of mosquito ecology and physiology than those from small cage colonies