Production of germ-free mosquitoes via transient colonisation allows stage-specific investigation of host-microbiota interactions.

The mosquito microbiota impacts the physiology of its host and is essential for normal larval development, thereby influencing transmission of vector-borne pathogens. Germ-free mosquitoes generated with current methods show larval stunting and developmental deficits. Therefore, functional studies of the mosquito microbiota have so far mostly been limited to antibiotic treatments of emerging adults. In this study, we introduce a method to produce germ-free Aedes aegypti mosquitoes. It is based on reversible colonisation with bacteria genetically modified to allow complete decolonisation at any developmental stage. We show that, unlike germ-free mosquitoes previously produced using sterile diets, reversibly colonised mosquitoes show no developmental retardation and reach the same size as control adults. This allows us to uncouple the study of the microbiota in larvae and adults. In adults, we detect no impact of bacterial colonisation on mosquito fecundity or longevity. In larvae, data from our transcriptome analysis and diet supplementation experiments following decolonisation suggest that bacteria support larval development by contributing to folate biosynthesis and by enhancing energy storage. Our study establishes a tool to study the microbiota in insects and deepens our knowledge on the metabolic contribution of bacteria to mosquito development.

Activity of additives and their effect in formulations of Metarhizium anisopliae s.l. IP 46 against Aedes aegypti adults and on post mortem conidiogenesis.

BACKGROUND: Oil formulations of entomopathogenic fungi have interest for biological mosquito control. OBJECTIVES: The activities of M. anisopliae s.l. IP 46 conidia were tested in Aedes aegypti adults either without any formulation or formulated with vegetable or mineral oil and in combination with diatomaceous earth. FINDINGS: IP 46 was highly active against adults, the vector of important arboviruses in the tropics and subtropics. At an exposure of adults to 3.3 × 107 conidia/cm2, values of lethal times TL50 and TL90 reached minimal 3.8 and 4.6 days, respectively, and lethal concentrations LC50 and LC90 were 2.7 × 105 and 2.4 × 106 conidia/cm2, respectively, after 10 days of exposure. Activity against adults was improved by diatomaceous earth (KeepDry® KD) combined with mineral oil (Naturol® N) or vegetable oil (Graxol® G). Additives KD or N separately (and G to a lesser extent) or in combination, KD + N and KD + G without conidia had also a clear adulticidal effect. Efficacy of conidia formulated or not with KD + N decreased somewhat at shorter exposure periods. Time of exposure (0.017, 12, 48, 72 or 120 h) of adults to KD and N or IP 46 or conidia and KD and N had no significant effect on mortality. M. anisopliae s.l. recycled on fungus-killed mosquitoes producing high quantities of new conidia regardless of the conidial concentrations or formulations tested. Additives tested had no clear effect on quantitative conidiogenesis on cadavers. MAIN CONCLUSIONS: Formulations of IP 46 conidia with mineral oil and diatomaceous earth represent a promising tool for the development of potent strategies of focal control of this important vector with entomopathogenic fungi.

A highly stable blood meal alternative for rearing Aedes and Anopheles mosquitoes.

We investigated alternatives to whole blood for blood feeding of mosquitoes with a focus on improved stability and compatibility with mass rearing programs. In contrast to whole blood, an artificial blood diet of ATP-supplemented plasma was effective in maintaining mosquito populations and was compatible with storage for extended periods refrigerated, frozen, and as a lyophilized powder. The plasma ATP diet supported rearing of both Anopheles and Aedes mosquitoes. It was also effective in rearing Wolbachia-infected Aedes mosquitoes, suggesting compatibility with vector control efforts.

Oviposition responses of Aedes mosquitoes to bacterial isolates from attractive bamboo infusions.

BACKGROUND: The mosquitoes Aedes aegypti and Aedes albopictus are vectors of pathogenic viruses that cause major human illnesses including dengue, yellow fever and chikungunya. Both mosquito species are expanding their geographic distributions and now occur worldwide in temperate and tropical climates. Collection of eggs in oviposition traps (ovitraps) is commonly used for monitoring and surveillance of container-inhabiting Aedes populations by public health agencies charged with managing mosquito-transmitted illness. Addition of an organic infusion in these traps increases the number of eggs deposited. Gravid females are guided to ovitraps by volatile chemicals produced from the breakdown of organic matter by microbes. METHODS: We previously isolated and cultured 14 species of bacteria from attractive experimental infusions, made from the senescent leaves of canebrake bamboo (Arundinaria gigantea). Cultures were grown for 24 h at 28 °C with constant shaking (120 rpm) and cell densities were determined with a hemocytometer. Behavioral responses to single bacterial isolates and to a mix of isolates at different cell densities were evaluated using two-choice sticky-screen bioassay methods with gravid Ae. aegypti and Ae. albopictus. RESULTS: In behavioral assays of a mix of 14 bacterial isolates, significantly greater attraction responses were exhibited by Ae. aegypti and Ae. albopictus to bacterial densities of 10(7) and 10(8) cells/mL than to the control medium. When we tested single bacterial isolates, seven isolates (B1, B2, B3, B5, B12, B13 and B14) were significantly attractive to Ae. aegypti, and six isolates (B1, B5, B7, B10, B13 and B14) significantly attracted Ae. albopictus. Among all the isolates tested at three different cell densities, bacterial isolates B1, B5, B13 and B14 were highly attractive to both Aedes species. CONCLUSIONS: Our results show that at specific cell densities, some bacteria significantly influence the attraction of gravid Ae. aegypti and Ae. albopictus females to potential oviposition sites. Attractive bacterial isolates, when formulated for sustained release of attractants, could be coupled with an ovitrap containing a toxicant to achieve area-wide management of Aedes mosquitoes.

Competition for amino acids between Wolbachia and the mosquito host, Aedes aegypti.

The endosymbiont Wolbachia represents a promising method of dengue control, as it reduces the ability of the primary vector, the mosquito Aedes aegypti, to transmit viruses. When mosquitoes infected with the virulent Wolbachia strain wMelPop are fed non-human blood, there is a drastic reduction in mosquito fecundity and egg viability. Wolbachia has a reduced genome and is clearly dependent on its host for a wide range of nutritional needs. The fitness defects seen in wMelPop-infected A. aegypti could be explained by competition between the mosquito and the symbiont for essential blood meal nutrients, the profiles of which are suboptimal in non-human blood. Here, we examine cholesterol and amino acids as candidate molecules for competition, as they have critical roles in egg structural development and are known to vary between blood sources. We found that Wolbachia infection reduces total cholesterol levels in mosquitoes by 15-25%. We then showed that cholesterol supplementation of a rat blood meal did not improve fecundity or egg viability deficits. Conversely, amino acid supplementation of sucrose before and after a sheep blood meal led to statistically significant increases in fecundity of approximately 15-20 eggs per female and egg viability of 30-40%. This mosquito system provides the first empirical evidence of competition between Wolbachia and a host over amino acids and may suggest a general feature of Wolbachia-insect associations. These competitive processes could affect many aspects of host physiology and potentially mosquito fitness, a key concern for Wolbachia-based mosquito biocontrol.

Trypsin inhibitor from Moringa oleifera flowers interferes with survival and development of Aedes aegypti larvae and kills bacteria inhabitant of larvae midgut.

Moringa oleifera flower extract, with trypsin inhibitor activity, is a larvicidal agent on Aedes aegypti. This work reports the isolation of trypsin inhibitor (M. oleifera flower trypsin inhibitor (MoFTI)) and its effect on A. aegypti egg hatching, viability of newly hatched larvae, survival of pupae, and growth of inhabitant bacteria from midgut of fourth-instar larvae (L4). MoFTI (K i, 2.4 µM), isolated by affinity chromatography on trypsin-agarose column, was an 18.2 kDa polypeptide on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Flower extract (at concentrations of 8.5-17.0 mg/mL) reduced egg hatchability while MoFTI (0.05-0.5 mg/mL) did not affect the hatching rate. Mortality of newly hatched larvae ranged from 3.5 to 19.1 % in the presence of the extract (4.0-17.0 mg/mL) and was also promoted by MoFTI (LC50, 0.3 mg/mL). After 72 h, larvae incubated with extract at 13.0 and 17.0 mg/mL were at stages L2 and L1, respectively, while in control they reached L3 instar. In the presence of MoFTI, at all concentrations tested, the larvae did not pass the first instar. Flower extract and MoFTI did not interfere on pupae survival. The extract and MoFTI inhibited the growth of L4 gut bacteria (minimum inhibitory concentrations of 3.47 and 0.031 mg/mL, respectively) but only the inhibitor showed bactericide effect (minimum bactericidal concentration of 1.0 mg/mL). The findings reported herein indicate that MoFTI constitutes a larvicidal principle from M. oleifera flowers against A. aegypti newly hatched larvae and is an antibacterial agent active against the microbiota from L4 gut.

Integration of botanical and bacterial insecticide against Aedes aegypti and Anopheles stephensi.

The present study evaluated the Orthosiphon thymiflorus leaf extract and the bacterial insecticide spinosad, testing the first to fourth instars larvae and pupae of two important vector mosquitoes, viz., Aedes aegypti, Anopheles stephensi. The fresh leaves of O. thymiflorus were washed thoroughly in tap water and shade-dried at room temperature (28 ± 2 °C) for 5 to 8 days. The air-dried materials were powdered separately using a commercial electrical blender. From the plants, 500 g powder was macerated with 1.5 L organic solvents of petroleum ether sequentially for a period of 72 h each and then filtered. The larval and pupal mortality was observed after 24 h of exposure; no mortality was observed in the control group. The first- to fourth-instar larvae and pupae of A. stephensi had values of LC(50) = 309.16, 337.58, 390.42, 429.68, and 513.34 ppm, and A. aegypti had values of LC(50) = 334.78, 366.45, 422.97, 467.94, and 54.02 ppm, respectively. Spinosad against the A. stephensi had values of LC(50) = 384.19, 433.39, 479.17, 519.79, and 572.63 ppm, and A. aegypti had values of LC(50) = 210.68, 241.20, 264.93, 283.27, and 305.85 ppm, respectively. Moreover, in combined treatment, the A. stephensi had values of LC(50) = 202.36, 224.76, 250.84, 288.05, and 324.05 ppm, and A. aegypti had values of LC(50) = 217.70, 246.04, 275.36, 315.29, and 353.80 ppm, respectively. Results showed that the leaf extract of O. thymiflorus and bacterial insecticide spinosad are promising as a good larvicidal and pupicidal against dengue vector, A. aegypti and malarial vector, A. stephensi. This is an ideal eco-friendly approach for the control of target species of vector control programs.

Toxicity of Bacillus thuringiensis subsp. israelensis to adult Aedes aegypti mosquitoes.

Adult female Aedes aegypti mosquitoes were killed by the parasporal crystals of Bacillus thuringiensis subsp. israelensis (ONR-60A) when the crystals were introduced into the insect midgut as an enema. The 50% lethal dose for intact parasporal crystals was 0.21 microgram/mg of mosquito (wet weight), and for solubilized crystals the 50% lethal dose was 0.04 microgram/mg. These values were compared with 50% lethal concentrations in a free-feeding larval mosquito bioassay of 0.018 and 1.28 microgram/ml for intact and solubilized crystals, respectively. Preparations from B. thuringiensis subsp. kurstaki were ineffective against both adult and larval mosquitoes. An adult mosquito bioassay is suggested as a direct means of screening potential mosquito control agents.