Effect of Microsporidia MB infection on the development and fitness of Anopheles arabiensis under different diet regimes.

BACKGROUND: Microsporidia MB (MB) is a naturally occurring symbiont of Anopheles and has recently been identified as having a potential to inhibit the transmission of Plasmodium in mosquitoes. MB intensity is high in mosquito gonads, with no fitness consequences for the mosquito, and is linked to horizontal (sexual) and vertical (transovarial) transmission from one mosquito to another. Maximising MB intensity and transmission is important for maintaining heavily infected mosquito colonies for experiments and ultimately for mosquito releases. We have investigated how diet affects the MB-Anopheles arabiensis symbiosis phenotypes, such as larval development and mortality, adult size and survival, as well as MB intensity in both larvae and adults. METHODS: F1 larvae of G0 females confirmed to be An. arabiensis and infected with MB were either combined (group lines [GLs]) or reared separately (isofemale lines [IMLs]) depending on the specific experiment. Four diet regimes (all mg/larva/day) were tested on F1 GLs: Tetramin 0.07, Tetramin 0.3, Gocat 0.3 and Cerelac 0.3. GLs reared on Tetramin 0.3 mg/larva/day were then fed either a 1% or 6% glucose diet to determine adult survival. Larvae of IMLs were fed Tetramin 0.07 mg and Tetramin 0.3 mg for larval experiments. The mosquitoes in the adult experiments with IMLs were reared on 1% or 6% glucose. RESULTS: Amongst the four larval diet regimes tested on An. arabiensis development in the presence of MB, the fastest larval development highest adult emergence, largest body size of mosquitoes, highest prevalence and highest density of MB occurred in those fed Tetramin 0.3 mg/larva/day. Although adult MB-positive mosquitoes fed on 6% glucose survived longer than MB-negative mosquitoes, there was no such effect for those fed on the 1% glucose diet. Development time, wing length and adult survival were not significantly different between MB-infected and uninfected An. arabiensis fed on the Tetramin 0.07 mg/larva/day diet, demonstrating that the MB-conferred fitness advantage was diet-dependent. CONCLUSIONS: Microsporidia MB does not adversely impact the development and fitness of An. arabiensis, even under limited dietary conditions. The diet regime of Tetramin 0.3 mg/larva/day + 6% glucose for adults is the superior diet for the mass rearing of MB-infected An. arabiensis mosquitoes. These results are important for rearing MB-infected An. arabiensis in the laboratory for experiments and the mass rearing required for field releases.

Efficacy of Metarhizium anisopliae, Isolate ICIPE 7, against Anopheles arabiensis, Glossina fuscipes, and Rhipicephalus spp.

Arthropod vectors are responsible for a multitude of human and animal diseases affecting poor communities in sub-Saharan Africa. Their control still relies on chemical agents, despite growing evidence of insecticide resistance and environmental health concerns. Biorational agents, such as the entomopathogenic fungus Metarhizium anisopliae, might be an alternative for vector control. Recently, the M. anisopliae isolate ICIPE 7 has been developed into a commercial product in Kenya for control of ticks on cattle. We were interested in assessing the potential of controlling not only ticks but also disease-transmitting mosquitoes and tsetse flies using cattle as blood hosts, with the aim of developing a product for integrated vector management. Laboratory bioassays were carried out with M. anisopliae, isolate ICIPE 7 and isolate ICIPE 30, to compare efficacy against laboratory-reared Anopheles arabiensis. ICIPE 7 was further tested against wild Glossina fuscipes and Rhipicephalus spp. Dose-response tests were implemented, period of mosquito exposure was evaluated for effects on time to death, and the number of spores attached to exposed vectors was assessed. Exposure to 109 spores/mL of ICIPE 7 for 10 min resulted in a similar mortality of An. arabiensis as exposure to ICIPE 30, albeit at a slower rate (12 vs. 8 days). The same ICIPE 7 concentration also resulted in mortalities of tsetse flies (LT50: 16 days), tick nymphs (LT50: 11 days), and adult ticks (LT50: 20 days). Mosquito mortality was dose-dependent, with decreasing LT50 of 8 days at a concentration of 106 spores/mL to 6 days at 1010 spores/mL. Exposure period did not modulate the outcome, 1 min of exposure still resulted in mortality, and spore attachment to vectors was dose-dependent. The laboratory bioassays confirmed that ICIPE 7 has the potential to infect and cause mortality to the three exposed arthropods, though at slower rate, thus requiring further validation under field conditions.

Willingness to accept and participate in a Microsporidia MB-based mosquito release strategy: a community-based rapid assessment in western Kenya.

BACKGROUND: Microsporidia MB, an endosymbiont naturally found in Anopheles mosquitoes inhibits transmission of Plasmodium and is a promising candidate for a transmission-blocking strategy that may involve mosquito release. A rapid assessment was carried out to develop insight into sociodemographic factors, public health concerns, and malaria awareness, management, and prevention practices with the willingness to accept and participate in Microsporidia MB-based transmission-blocking strategy to develop an informed stakeholder engagement process. METHODS: The assessment consisted of a survey conducted in two communities in western Kenya that involved administering a questionnaire consisting of structured, semi-structured, and open questions to 8108 household heads. RESULTS: There was an overall high level of willingness to accept (81%) and participate in the implementation of the strategy (96%). Although the willingness to accept was similar in both communities, Ombeyi community was more willing to participate (OR 22, 95% CI 13-36). Women were less willing to accept (OR 0.8, 95% CI 0.7-0.9) compared to men due to fear of increased mosquito bites near homes. Household heads with incomplete primary education were more willing to accept (OR 1.6, 95% CI 01.2-2.2) compared to those educated to primary level or higher. Perceiving malaria as a moderate or low public health issue was also associated with a lower willingness to accept and participate. Experience of > 3 malaria cases in the family over the last six months and knowledge that malaria is transmitted by only mosquito bites, increased the willingness to accept but reduced the willingness to participate. Awareness of malaria control methods based on mosquitoes that cannot transmit malaria increases the willingness to participate. CONCLUSION: The study showed a high level of willingness to accept and participate in a Microsporidia MB-based strategy in the community, which is influenced by several factors such as community, disease risk perception, gender, education level, knowledge, and experience of malaria. Further research will need to focus on understanding the concerns of women, educated, and employed community members, and factors that contribute to the lower disease risk perception. This improved understanding will lead to the development of an effective communication strategy.

Evaluation of the solar-powered Silver Bullet 2.1 (Lumin 8) light trap for sampling malaria vectors in western Kenya.

BACKGROUND: Centers for Disease Control and Prevention (CDC) light traps are widely used for sampling mosquitoes. However, this trap, manufactured in the USA, poses challenges for use in sub-Saharan Africa due to procurement costs and shipping time. Traps that are equally efficient than the CDC light trap, but which are amenable for use in remote African settings and made in Africa, are desirable to improve local vector surveillance. This study evaluated a novel solar-powered light trap made in South Africa (Silver Bullet trap; SB), for its efficiency in malaria vector sampling in western Kenya. METHODS: Large cage (173.7 m3) experiments and field evaluations were conducted to compare the CDC-incandescent light trap (CDC-iLT), CDC-UV fluorescent tube light trap (CDC-UV), SB with white diodes (SB-White) and SB with UV diodes (SB-UV) for sampling Anopheles mosquitoes. Field assessments were done indoors and outdoors following a Latin square design. The wavelengths and absolute spectral irradiance of traps were compared using spectrometry. RESULTS: The odds of catching a released Anopheles in the large cage experiments with the SB-UV under ambient conditions in the presence of a CDC-iLT in the same system was three times higher than what would have been expected when the two traps were equally attractive (odds ratio (OR) 3.2, 95% confidence interval CI 2.8-3.7, P < 0.01)). However, when the white light diode was used in the SB trap, it could not compete with the CDC-iLT (OR 0.56, 95% CI 0.48-0.66, p < 0.01) when the two traps were provided as choices in a closed system. In the field, the CDC and Silver Bullet traps were equally effective in mosquito sampling. Irrespective of manufacturer, traps emitting UV light performed better than white or incandescent light for indoor sampling, collecting two times more Anopheles funestus sensu lato (s.l.) (RR 2.5; 95% CI 1.7-3.8) and Anopheles gambiae s.l. (RR 2.5; 95% 1.7-3.6). Outdoor collections were lower than indoor collections and similar for all light sources and traps. CONCLUSIONS: The solar-powered SB trap compared well with the CDC trap in the field and presents a promising new surveillance device especially when charging on mains electricity is challenging in remote settings.

Genetics and immunity of Anopheles response to the entomopathogenic fungus Metarhizium anisopliae overlap with immunity to Plasmodium.

Entomopathogenic fungi have been explored as a potential biopesticide to counteract the insecticide resistance issue in mosquitoes. However, little is known about the possibility that genetic resistance to fungal biopesticides could evolve in mosquito populations. Here, we detected an important genetic component underlying Anopheles coluzzii survival after exposure to the entomopathogenic fungus Metarhizium anisopliae. A familiality study detected variation for survival among wild mosquito isofemale pedigrees, and genetic mapping identified two loci that significantly influence mosquito survival after fungus exposure. One locus overlaps with a previously reported locus for Anopheles susceptibility to the human malaria parasite Plasmodium falciparum. Candidate gene studies revealed that two LRR proteins encoded by APL1C and LRIM1 genes in this newly mapped locus are required for protection of female A. coluzzii from M. anisopliae, as is the complement-like factor Tep1. These results indicate that natural Anopheles populations already segregate frequent genetic variation for differential mosquito survival after fungal challenge and suggest a similarity in Anopheles protective responses against fungus and Plasmodium. However, this immune similarity raises the possibility that fungus-resistant mosquitoes could also display enhanced resistance to Plasmodium, suggesting an advantage of selecting for fungus resistance in vector populations to promote naturally diminished malaria vector competence.

Grass-like plants release general volatile cues attractive for gravid Anopheles gambiae sensu stricto mosquitoes.

BACKGROUND: Understanding the ecology and behaviour of disease vectors, including the olfactory cues used to orient and select hosts and egg-laying sites, are essential for the development of novel, insecticide-free control tools. Selected graminoid plants have been shown to release volatile chemicals attracting malaria vectors; however, whether the attraction is selective to individual plants or more general across genera and families is still unclear. METHODS: To contribute to the current evidence, we implemented bioassays in two-port airflow olfactometers and in large field cages with four live graminoid plant species commonly found associated with malaria vector breeding sites in western Kenya: Cyperus rotundus and C. exaltatus of the Cyperaceae family, and Panicum repens and Cynodon dactylon of the Poaceae family. Additionally, we tested one Poaceae species, Cenchrus setaceus, not usually associated with water. The volatile compounds released in the headspace of the plants were identified using gas chromatography/mass spectrometry. RESULTS: All five plants attracted gravid vectors, with the odds of a mosquito orienting towards the choice-chamber with the plant in an olfactometer being 2-5 times higher than when no plant was present. This attraction was maintained when tested with free-flying mosquitoes over a longer distance in large field cages, though at lower strength, with the odds of attracting a female 1.5-2.5 times higher when live plants were present than when only water was present in the trap. Cyperus rotundus, previously implicated in connection with an oviposition attractant, consistently elicited the strongest response from gravid vectors. Volatiles regularly detected were limonene, ß-pinene, ß-elemene and ß-caryophyllene, among other common plant compounds previously described in association with odour-orientation of gravid and unfed malaria vectors. CONCLUSIONS: The present study confirms that gravid Anopheles gambiae sensu stricto use chemical cues released from graminoid plants to orientate. These cues are released from a variety of graminoid plant species in both the Cyperaceae and Poaceae family. Given the general nature of these cues, it appears unlikely that they are exclusively used for the location of suitable oviposition sites. The utilization of these chemical cues for attract-and-kill trapping strategies must be explored under natural conditions to investigate their efficiency when in competition with complex interacting natural cues.

Horizontal Transmission of the Symbiont Microsporidia MB in Anopheles arabiensis.

The recently discovered Anopheles symbiont, Microsporidia MB, has a strong malaria transmission-blocking phenotype in Anopheles arabiensis, the predominant Anopheles gambiae species complex member in many active transmission areas in eastern Africa. The ability of Microsporidia MB to block Plasmodium transmission together with vertical transmission and avirulence makes it a candidate for the development of a symbiont-based malaria transmission blocking strategy. We investigate the characteristics and efficiencies of Microsporidia MB transmission between An. arabiensis mosquitoes. We show that Microsporidia MB is not transmitted between larvae but is effectively transmitted horizontally between adult mosquitoes. Notably, Microsporidia MB was only found to be transmitted between male and female An. arabiensis, suggesting sexual horizontal transmission. In addition, Microsporidia MB cells were observed infecting the An. arabiensis ejaculatory duct. Female An. arabiensis that acquire Microsporidia MB horizontally are able to transmit the symbiont vertically to their offspring. We also investigate the possibility that Microsporidia MB can infect alternate hosts that live in the same habitats as their An. arabiensis hosts, but find no other non-anopheline hosts. Notably, Microsporidia MB infections were found in another primary malaria African vector, Anopheles funestus s.s. The finding that Microsporidia MB can be transmitted horizontally is relevant for the development of dissemination strategies to control malaria that are based on the targeted release of Microsporidia MB infected Anopheles mosquitoes.

Comprehensive Ecological and Geographic Characterization of Eukaryotic and Prokaryotic Microbiomes in African Anopheles.

Exposure of mosquitoes to numerous eukaryotic and prokaryotic microbes in their associated microbiomes has probably helped drive the evolution of the innate immune system. To our knowledge, a metagenomic catalog of the eukaryotic microbiome has not been reported from any insect. Here we employ a novel approach to preferentially deplete host 18S ribosomal RNA gene amplicons to reveal the composition of the eukaryotic microbial communities of Anopheles larvae sampled in Kenya, Burkina Faso and Republic of Guinea (Conakry). We identified 453 eukaryotic operational taxonomic units (OTUs) associated with Anopheles larvae in nature, but an average of 45% of the 18S rRNA sequences clustered into OTUs that lacked a taxonomic assignment in the Silva database. Thus, the Anopheles microbiome contains a striking proportion of novel eukaryotic taxa. Using sequence similarity matching and de novo phylogenetic placement, the fraction of unassigned sequences was reduced to an average of 4%, and many unclassified OTUs were assigned as relatives of known taxa. A novel taxon of the genus Ophryocystis in the phylum Apicomplexa (which also includes Plasmodium) is widespread in Anopheles larvae from East and West Africa. Notably, Ophryocystis is present at fluctuating abundance among larval breeding sites, consistent with the expected pattern of an epidemic pathogen. Species richness of the eukaryotic microbiome was not significantly different across sites from East to West Africa, while species richness of the prokaryotic microbiome was significantly lower in West Africa. Laboratory colonies of Anopheles coluzzii harbor 26 eukaryotic OTUs, of which 38% (n = 10) are shared with wild populations, while 16 OTUs are unique to the laboratory colonies. Genetically distinct An. coluzzii colonies co-housed in the same facility maintain different prokaryotic microbiome profiles, suggesting a persistent host genetic influence on microbiome composition. These results provide a foundation to understand the role of the Anopheles eukaryotic microbiome in vector immunity and pathogen transmission. We hypothesize that prevalent apicomplexans such as Ophryocystis associated with Anopheles could induce interference or competition against Plasmodium within the vector. This and other members of the eukaryotic microbiome may offer candidates for new vector control tools.

A cross-sectional observational study investigating the association between sedges (swamp grasses, Cyperaceae) and the prevalence of immature malaria vectors in aquatic habitats along the shore of Lake Victoria, western Kenya.

Background: Strategies that involve manipulations of the odour-orientation of gravid malaria vectors could lead to novel attract-and-kill interventions. Recent work has highlighted the potential involvement of graminoid plants in luring vectors to oviposition sites. This study aimed to analyse the association between water-indicating graminoid plants (Cyperaceae, sedges), other abiotic and biotic factors and the presence and abundance of early instar Anopheles larvae in aquatic habitats as a proxy indicator for oviposition. Methods: A cross-sectional survey of 110 aquatic habitats along the shores of Lake Victoria was done during the rainy season. Habitats were sampled for mosquito larvae using the sweep-net method and habitat characteristics recorded. Results: Anopheles arabiensis was the dominant species identified from aquatic habitats. Larvae of the secondary malaria vectors such as Anopheles coustani, An. rufipes and An. maculipalpis were found only in habitats covered with graminoids, whereas An. arabiensis, An. ziemanni and An. pharoensis were found in both habitats with and without graminoid plants. The hypothesis that sedges might be positively associated with the presence and abundance of early instar Anopheles larvae could not be confirmed. The dominant graminoid plants in the habitats were Panicum repens, Cynodon dactylon in the Poaceae family and Cyperus rotundus in the Cyperaceae family. All of these habitats supported abundant immature vector populations. The presence of early instar larvae was significantly and positively associated with swamp habitat types (OR=22, 95% CI=6-86, P<0.001) and abundance of late Anopheles larvae (OR=359, CI=33-3941, P<0.001), whilst the association was negative with tadpole presence (OR=0.1, CI=0.0.01-0.5, P=0.008). Conclusions: Early instar malaria vectors were abundant in habitats densely vegetated with graminoid plants in the study area but specific preference for any of the graminoids could not be detected. In search for oviposition cues, it might be useful to screen for chemical volatiles released from all dominant plant species.

Direct and indirect effects of predation and parasitism on the Anopheles gambiae mosquito.

BACKGROUND: A good understanding of mosquito ecology is imperative for integrated vector control of malaria. In breeding sites, Anopheles larvae are concurrently exposed to predators and parasites. However, to our knowledge, there is no study on combined effects of predators and parasites on development and survival of larvae and their carry-over effects on adult survivorship and susceptibility to further parasite infection. METHODS: This study focused on effects of the nymphs of the dragonfly Pantala flavescens and the parasitic fungus Beauveria bassiana on Anopheles gambiae, to determine: predation efficacy of nymphs against An. gambiae larvae; development rate of An. gambiae larvae in the presence of one, two or four constrained nymphs; efficacy of B. bassiana against An. gambiae larvae at doses of 3, 6 and 12 mg; and survival of adult mosquitoes exposed to B. bassiana, following pre-exposure to a constrained predator and/or parasite at the larval stage. The experiments consisted of survival bioassays quantified as pupation day, or dead larvae and/or adults. RESULTS: Nymphs had an average predation efficacy of 88.3% (95% CI: 87.5-89.1) at 24 hours, against An. gambiae larvae. The presence of one or two nymphs reduced development time of larvae by 0.65 and 0.35 days, respectively. However, development time of larvae exposed to four nymphs was similar to the control larvae. Larvae exposed to 3, 6 and 12 mg of B. bassiana were 2.0, 2.5 and 3.5 times more likely to die, respectively, compared to control larvae. Adults not pre-exposed, those pre-exposed to predator, parasite, or both were 45.8, 67.4, 50.9 and 112.0 times more likely to die, respectively, compared to control that were unexposed to predator or parasite, at larval and adult stage. CONCLUSIONS: This study shows that both predator and parasite can reduce larval population of An. gambiae, and presence of predator cues decreases development time in breeding sites, as well as, increases the susceptibility of emerging adult to fungus. Predator and parasite both have an additive effect on survival of adults exposed to B. bassiana. Field studies are required for an in-depth understanding of predator and parasite influence on mosquito development time, survival and susceptibility in nature.

The Anopheles gambiae 2La chromosome inversion is associated with susceptibility to Plasmodium falciparum in Africa.

Chromosome inversions suppress genetic recombination and establish co-adapted gene complexes, or supergenes. The 2La inversion is a widespread polymorphism in the Anopheles gambiae species complex, the major African mosquito vectors of human malaria. Here we show that alleles of the 2La inversion are associated with natural malaria infection levels in wild-captured vectors from West and East Africa. Mosquitoes carrying the more-susceptible allele (2L+a) are also behaviorally less likely to be found inside houses. Vector control tools that target indoor-resting mosquitoes, such as bednets and insecticides, are currently the cornerstone of malaria control in Africa. Populations with high levels of the 2L+a allele may form reservoirs of persistent outdoor malaria transmission requiring novel measures for surveillance and control. The 2La inversion is a major and previously unappreciated component of the natural malaria transmission system in Africa, influencing both malaria susceptibility and vector behavior.

Fitness consequences of larval exposure to Beauveria bassiana on adults of the malaria vector Anopheles stephensi.

Entomopathogenic fungi have shown to be effective in biological control of both larval and adult stages of malaria mosquitoes. However, a small fraction of mosquitoes is still able to emerge after treatment with fungus during the larval stage. It remains unclear whether fitness of these adults is affected by the treatment during the larval stage and whether they are still susceptible for another treatment during the adult stage. Therefore, we tested the effects of larval exposure to the entomopathogenic fungus Beauveria bassiana on fitness of surviving Anopheles stephensi females. Furthermore, we tested whether larval exposed females were still susceptible to re-exposure to the fungus during the adult stage. Sex ratio, survival and reproductive success were compared between non-exposed and larval exposed A. stephensi. Comparisons were also made between survival of non-exposed and larval exposed females that were re-exposed to B. bassiana during the adult stage. Larval treatment did not affect sex ratio of emerging mosquitoes. Larval exposed females that were infected died significantly faster and laid equal numbers of eggs from which equal numbers of larvae hatched, compared to non-exposed females. Larval exposed females that were uninfected had equal survival, but laid a significantly larger number of eggs from which a significantly higher number of larvae hatched, compared to non-exposed females. Larval exposed females which were re-exposed to B. bassiana during the adult stage had equal survival as females exposed only during the adult stage. Our results suggest that individual consequences for fitness of larval exposed females depended on whether a fungal infection was acquired during the larval stage. Larval exposed females remained susceptible to re-exposure with B. bassiana during the adult stage, indicating that larval and adult control of malaria mosquitoes with EF are compatible.

Efficacy of aquatain, a monomolecular film, for the control of malaria vectors in rice paddies.

BACKGROUND: Rice paddies harbour a large variety of organisms including larvae of malaria mosquitoes. These paddies are challenging for mosquito control because their large size, slurry and vegetation make it difficult to effectively apply a control agent. Aquatain, a monomolecular surface film, can be considered a suitable mosquito control agent for such breeding habitats due to its physical properties. The properties allow Aquatain to self-spread over a water surface and affect multiple stages of the mosquito life cycle. METHODOLOGY/PRINCIPAL FINDINGS: A trial based on a pre-test/post-test control group design evaluated the potential of Aquatain as a mosquito control agent at Ahero rice irrigation scheme in Kenya. After Aquatain application at a dose of 2 ml/m(2) on rice paddies, early stage anopheline larvae were reduced by 36%, and late stage anopheline larvae by 16%. However, even at a lower dose of 1 ml/m(2) there was a 93.2% reduction in emergence of anopheline adults and 69.5% reduction in emergence of culicine adults. No pupation was observed in treated buckets that were part of a field bio-assay carried out parallel to the trial. Aquatain application saved nearly 1.7 L of water in six days from a water surface of 0.2 m(2) under field conditions. Aquatain had no negative effect on rice plants as well as on a variety of non-target organisms, except backswimmers. CONCLUSIONS/SIGNIFICANCE: We demonstrated that Aquatain is an effective agent for the control of anopheline and culicine mosquitoes in irrigated rice paddies. The agent reduced densities of aquatic larval stages and, more importantly, strongly impacted the emergence of adult mosquitoes. Aquatain also reduced water loss due to evaporation. No negative impacts were found on either abundance of non-target organisms, or growth and development of rice plants. Aquatain, therefore, appears a suitable mosquito control tool for use in rice agro-ecosystems.

Development of Metarhizium anisopliae and Beauveria bassiana formulations for control of malaria mosquito larvae.

BACKGROUND: The entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana have demonstrated effectiveness against anopheline larvae in the laboratory. However, utilising these fungi for the control of anopheline larvae under field conditions, relies on development of effective means of application as well as reducing their sensitivity to UV radiation, high temperatures and the inevitable contact with water. This study was conducted to develop formulations that facilitate the application of Metarhizium anisopliae and Beauveria bassiana spores for the control of anopheline larvae, and also improve their persistence under field conditions. METHODS: Laboratory bioassays were conducted to test the ability of aqueous (0.1% Tween 80), dry (organic and inorganic) and oil (mineral and synthetic) formulations to facilitate the spread of fungal spores over the water surface and improve the efficacy of formulated spores against anopheline larvae as well as improve spore survival after application. Field bioassays were then carried out to test the efficacy of the most promising formulation under field conditions in western Kenya. RESULTS: When formulated in a synthetic oil (ShellSol T), fungal spores of both Metarhizium anisopliae and Beauveria bassiana were easy to mix and apply to the water surface. This formulation was more effective against anopheline larvae than 0.1% Tween 80, dry powders or mineral oil formulations. ShellSol T also improved the persistence of fungal spores after application to the water. Under field conditions in Kenya, the percentage pupation of An. gambiae was significantly reduced by 39 - 50% by the ShellSol T-formulated Metarhizium anisopliae and Beauveria bassiana spores as compared to the effects of the application of unformulated spores. CONCLUSIONS: ShellSol T is an effective carrier for fungal spores when targeting anopheline larvae under both laboratory and field conditions. Entomopathogenic fungi formulated with a suitable carrier are a promising tool for control of larval populations of malaria mosquitoes. Additional studies are required to identify the best delivery method (where, when and how) to make use of the entomopathogenic potential of these fungi against anopheline larvae.

Factors affecting fungus-induced larval mortality in Anopheles gambiae and Anopheles stephensi.

BACKGROUND: Entomopathogenic fungi have shown great potential for the control of adult malaria vectors. However, their ability to control aquatic stages of anopheline vectors remains largely unexplored. Therefore, how larval characteristics (Anopheles species, age and larval density), fungus (species and concentration) and environmental effects (exposure duration and food availability) influence larval mortality caused by fungus, was studied. METHODS: Laboratory bioassays were performed on the larval stages of Anopheles gambiae and Anopheles stephensi with spores of two fungus species, Metarhizium anisopliae and Beauveria bassiana. For various larval and fungal characteristics and environmental effects the time to death was determined and survival curves established. These curves were compared by Kaplan Meier and Cox regression analyses. RESULTS: Beauveria bassiana and Metarhizium anisopliae caused high mortality of An. gambiae and An. stephensi larvae. However, Beauveria bassiana was less effective (Hazard ratio (HR) <1) compared to Metarhizium anisopliae. Anopheles stephensi and An. gambiae were equally susceptible to each fungus. Older larvae were less likely to die than young larvae (HR < 1). The effect of increase in fungus concentration on larval mortality was influenced by spore clumping. One day exposure to fungal spores was found to be equally effective as seven days exposure. In different exposure time treatments 0 - 4.9% of the total larvae, exposed to fungus, showed infection at either the pupal or adult stage. Mortality rate increased with increasing larval density and amount of available food. CONCLUSIONS: This study shows that both fungus species have potential to kill mosquitoes in the larval stage, and that mortality rate depends on fungus species itself, larval stage targeted, larval density and amount of nutrients available to the larvae. Increasing the concentration of fungal spores or reducing the exposure time to spores did not show a proportional increase and decrease in mortality rate, respectively, because the spores clumped together. As a result spores did not provide uniform coverage over space and time. It is, therefore, necessary to develop a formulation that allows the spores to spread over the water surface. Apart from formulation appropriate delivery methods are also necessary to avoid exposing non-target organisms to fungus.

Efficacy of Aquatain, a monomolecular surface film, against the malaria vectors Anopheles stephensi and An. gambiae s.s. in the laboratory.

Monomolecular films are used for mosquito control because of their asphyxiating effect on larvae and pupae. Compared with other films, Aquatain mosquito formulation (AMF) has an improved spreading ability and flexibility on a water surface. In the laboratory, AMF showed larvicidal, pupicidal, and oviposition deterrent effects against the malaria vectors Anopheles stephensi and An. gambiae. At a dose of 1 mL/m(2), the median lethal time to death for late larval instars was 3.02 (95% confidence interval [CI] = 2.76-3.25) and 0.98 (95% CI = 0.75-1.20) days for An. stephensi and An. gambiae, respectively. None of the treated larvae pupated. Pupal mortality reached 100% within two hours for both species. AMF repelled gravid females from ovipositing in treated oviposition cups. Without the choice of an untreated cup, the lowered water surface tension caused most females to drown while attempting to oviposit. This physical control method has the potential to become a safe, cost-effective, and resistance-proof malaria vector control tool.