A synthetic lure for Anopheles gambiae (Diptera: Culicidae) based on the attractive plant Parthenium hysterophorus.

Sugar is the sole diet for male mosquitoes and a complementary meal for females. Searching for natural sources of sugar is mediated by semiochemicals. Floral nectars, extra floral nectaries, damaged tissues of plants and rotten fruits are the most common sources of sugar in nature. I provide laboratory evidence of the high attraction of Parthenium hysterophorus L., a weed that grows in tropical climates, to Anopheles gambiae Giles. This study has tried to identify the chemicals which might be involved in the chemical attraction of A. gambiae to this plant. Using quantitative GC-MS analysis, α-pinene, camphene, 1-octen-3-ol, ß-pinene, cis-ß-ocimene, bornyl acetate, α-caryophyllene, hexadecanoic acid, and α-linolenic acid were identified as the main constituents of P. hysterophorus volatiles. Successive olfactory assays helped a better understanding of the more attractive chemicals of P. hysterophorus to A. gambiae which was the basis for testing a possible synthetic blend. Olfactory experiments proved this synthetic blend to be as attractive as Parthenium intact plants for A. gambiae. A minimal blend, consisting of only α-pinene, camphene, and cis-ß-ocimene, was also produced and laboratory experiments indicated its relative attraction for A. gambiae. This blend can be tested in the attractive toxic sugar bait stations for sampling, surveillance, or control programs of mosquitoes in tropical Africa, where A. gambiae sensu stricto transfer malaria among residents.

Deterrent Effects of Glyphosate on Oviposition and Larval Development of Culex quinquefasciatus.

Glyphosate is one of the most common herbicides used to control weeds in the USA. This herbicide can impact the mosquito life cycle through being carried to mosquito habitats by runoff. This study investigates the effects of glyphosate on the larval development and oviposition of a laboratory colony of Culex quinquefasciatus. Our experiments indicated that a concentration of 1 g/liter glyphosate was lethal to larval instars or the larvae impacted were either unable to molt to pupae or never emerged as adults. Larval instars exposed to 0.5 g/liter glyphosate experienced a similar impact; however, the larval stage was extended and pupation was considerably delayed. Mosquitoes oviposited in 0.5 g/liter glyphosate laid the same number of egg rafts as in water, but a considerably lower number of eggs exposed to glyphosate hatched as first instars. If gravid females laid their eggs in 1 g/liter glyphosate versus water, the difference between the 2 groups would be statistically significant and a very low number of eggs exposed to glyphosate could ever hatch into larvae. In an oviposition assay carried out in a cage with 3 choices of water, 0.5 g/liter, and 1 g/liter glyphosate, a higher number of egg rafts were laid in water, followed by 0.5 g/liter and 1 g/liter glyphosate concentrations, respectively. Our study indicated such a severe effect of glyphosate on all immature stages of Cx. quinquefasciatus that no adult could ever emerge.

Toxic Effect of Blood Feeding in Male Mosquitoes.

Blood- and sugar feeding of female mosquitoes has been frequently observed in the laboratory and in the field, but only sugar feeding of males has been reported. Here, we describe for the first time that Culex quinquefasciatus males feed on blood as well. Blood feeding easily happened on a blood-soaked cotton roll and, to a lesser extent, through a thin artificial layer. Mating history of a male specimen does not affect his blood feeding behavior. Male mosquitoes feed on blood even when they have a readily available sugar source. Nevertheless, feeding on blood reduces the survival rate of males to just a few days, as compared to more than a month for mosquitoes fed only on sugar. Comparing survival of male mosquitoes fed on blood only, sugar only, and a combination of both clearly demonstrated that mortality is not affected by malnutrition (reduced sugar levels), but rather due to ingested blood. On average male mosquitoes ingested ca. 0.5 µl of blood, i.e., about 10% of the amount of blood ingested by an engorged female. Although this unexpected observation of blood feeding in the laboratory by male mosquitoes is interesting, structural impairment prevents male feeding on vertebrate blood. In agreement with the literature, male and female proboscises and stylets were in general of similar size, but male mandibles were significantly shorter than female counterparts, thus explaining their inability to pierce through skin layers.

Orientation of Anopheles gambiae (Diptera: Culicidae) to Plant-Host Volatiles in a Novel Diffusion-Cage Olfactometer.

A novel diffusion-cage olfactometer tested the responses of Anopheles gambiae Giles to plant volatiles. Green-leaf volatiles are often released from cut or injured plant tissue and may alter the headspace of plants used in olfactometer assays. The diffusion-cage olfactometer is designed for use with whole, intact plants, hence giving a more realistic behavioral assay. Its simple plastic construction, ease of assembly, and accommodation to whole plants makes it a useful tool for measuring mosquito orientation to plant volatiles within large enclosures. We compared its performance to that of the more commonly used T-tube wind-tunnel olfactometer, by testing the orientation of mosquitoes to volatiles of a few prevalent plants of eastern Africa reportedly utilized by An. gambiae for sugar: Parthenium hysterophorus (Asteraceae), Ricinus communis (Euphorbiaceae), Lantana camara (Verbenaceae), and Senna occidentalis (Fabaceae). Results indicate that the diffusion-cage olfactometer is an effective alternative to conventional wind-tunnel olfactometers, to test mosquito orientation to plant volatiles under seminatural conditions.

Evaluation of Bacillus thuringiensis israelensis as a Control Agent for Adult Anopheles gambiae.

Unlike the application of Bacillus thuringiensis israelensis (Bti) for the control of larval mosquitoes, studies of its effects on adults, for its possible use in attractive toxic sugar baits, have resulted in conflicting results. Five species have shown a decrease in adult survival due to Bti ingestion, whereas adults of Anopheles arabiensis have not. We sought to determine if ingestion of Bti by adults of Anopheles gambiae, a sibling species of An. arabiensis, increases their mortality. Laboratory-reared adults were provided continuously from emergence with water only, a sucrose solution, or a Bti suspension in sucrose solution. After 3 days, the Bti suspension was replaced with untainted sucrose solution. The mosquitoes with only water were all dead by day 3. The survivorships of those in the sucrose and sucrose-Bti treatments were insignificantly different, both with an LT50 (Lethal Time, time until 50% of individuals died) of 25 days. The results support the conclusion that adult survivorship of An. gambiae-complex members is unaffected by the ingestion of Bti in sugar meals.

Olfactory basis of floral preference of the malaria vector Anopheles gambiae (Diptera: Culicidae) among common African plants.

Mosquitoes of both sexes feed on plants to obtain sugar. Nocturnal species probably locate the plants primarily by their volatile semiochemicals that also form the basis for the mosquitoes' innate plant-species preferences. To evaluate these olfactory preferences quantitatively, we used a two-choice wind-tunnel olfactometer to measure the upwind orientation of Anopheles gambiae Giles, an important vector of malaria in equatorial Africa, toward odor plumes produced by nine plant species common where this mosquito occurs. These plants are reported to induce feeding behaviors in An. gambiae and to produce floral or extrafloral nectar. Results presented here demonstrated that the volatiles of S. didymobotrya, P. hysterophorus, S. occidentalis, and L. camara, in descending order of numbers of mosquitoes responding, were all attractive, compared to a control plant species, whereas D. stramonium, R. communis, S. bicapsularis, T. stans, and T. diversifolia were not. As expected, chromatographic analysis of the headspace of attractive plants whose volatiles were captured by stir-bar sorptive extraction revealed a wide range of compounds, primarily terpenoids. Once their bioactivity and attractiveness for An. gambiae, alone and in blends, has been firmly established, some of these semiochemicals may have applications in population sampling and control.

Rapid discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) analysis.

There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software.