Susceptibility of adult Aedes aegypti (Diptera: Culicidae) to infection by Metarhizium anisopliae and Beauveria bassiana: prospects for Dengue vector control

Dengue fever vectored by the mosquito Aedes aegypti is one of the most rapidly spreading insect-borne diseases, stimulating the search for alternatives to current control methods. Screening assays using a range of Metarhizium anisopliae and Beauveria bassiana isolates were performed against adult female Ae. aegypti. Four virulent isolates were selected for detailed study. Adult female mosquitoes were exposed to supports previously inoculated with fungal suspensions. Fungal isolates were suspended in Tween 80+8% vegetable oil. The isolates caused between 70 and 89% mortality as a result of fungal infection over the 7-day test period. Mean survival times varied between 3 and 5 days for treated insects, whilst control survival exceeded 40 days. The most promising isolate, M. anisopliae LPP133, based not only on virulence but facility for mass production, was used for lethal exposure time determinations. An exposure time of only 3.5 h was necessary to cause 50% mortality. Large cage trails were also carried out and mean survival time of insects exposed to fungus impregnated black cloths was significantly reduced. These results show that entomopathogenic fungi could be promising biological control agents for use against adult Ae. aegypti, by inoculating fungi onto surfaces on which the mosquitoes tend to rest. The subsequent mortality caused by the fungi could potentially reduce the populations of this insect thus reducing the incidence of Dengue.     

The potential of Metarhizium anisopliae and Beauveria bassiana isolates for the control of Aedes aegypti (Diptera: Culicidae) larvae

Fungal isolates were screened against Aedes aegypti larvae. Exposure of larvae to conidial suspensions resulted in 6–90% mortality. An inoculum persistence assay using one of the most virulent isolates showed an approximate half-life of 10 days in water containers. Not all larvae surviving to form pupae resulted in adult emergence.     

Polymorphic microsatellite markers for studies of Aedes aegypti (Diptera: Culicidae), the vector of dengue and yellow fever

A significant challenge to population genetic studies of the dengue vector, Aedes aegypti, has been the lack of polymorphic microsatellite loci. In an effort to develop useful markers, we evaluated the genetic variation at 17 microsatellite loci identified in the A. aegypti genome. Nine loci with at least five alleles were identified in field‐collected specimens from Thailand. An additional two loci carried five alleles if samples from an A. aegypti laboratory colony were included. Our results greatly increase the number of highly variable markers available for the study of the genetics and the population structure of this medically important species.     

Evaluation of entomopathogenic fungi as potential biological control agents of the dengue mosquito,Aedes aegypti (Diptera: Culicidae)

Dengue is a global health concern. Growing insecticide resistance in the primary mosquito vector, Aedes aegypti, limits the effectiveness of vector control, so alternative tools are urgently needed. One approach is the use of biopesticides comprising entomopathogenic fungi, e.g., Beauveria bassiana and Metarhizium anisopliae. These fungi may decrease disease transmission by reducing mosquito vector longevity and also occur worldwide, although many isolates have not been tested for virulence against mosquitoes. Ninety-three isolates of entomopathogenic fungi representing six species (B. bassiana, M. anisopliae, Isaria fumosorosea, I. farinosa, I. flavovirescens, and Lecanicillium spp.) were screened as potential biological control agents of Aedes aegypti. A hierarchical, multi-criteria experimental design was undertaken to find suitable isolates. Initial screening was performed via in vitro assays measuring radial growth and spore persistence, eliminating isolates with poor growth or viability on nutrient-rich substrate. Subsequent measurements of spore persistence revealed that only nine of 30 strains tested had half-lives exceeding 3 weeks. Ten isolates were chosen for in vivo bioassays against adult Ae. aegypti. From these assays, two Australian isolates of B. bassiana, FI-277 and FI-278, appeared to be most promising. Both isolates were shown to be virulent against Ae. aegypti at 20, 26, and 32°C. Spreading spores manually onto substrate was found to be more efficacious than spraying. Ae. aegypti infected by manually-spread spores on cotton substrate were found to have an LT₅₀ of 3.7±0.3 days. These characteristics suggest that FI-277 has promise as a dengue mosquito biocontrol agent, either alone or combined with conventional chemical insecticides.     

Feeding response of Aedes aegypti and Anopheles dirus (Diptera: Culicidae) using out‐of‐date human blood in a membrane feeding apparatus

The colonization of Aedes aegypti and Anopheles dirus was performed using out‐of‐date human blood from a blood bank as a nutritional supply dispensed from a common artificial feeder. Preserved human blood was collected and used for feeding on days 5, 15, and 25 after date of expiration and dispensed from a common artificial feeder to rear the mosquitoes. Ae. aegypti had a feeding rate of 78.7, 62, and 18% at the respective intervals while An. dirus had a rate of 80, 56.8, and 7.3% on the same respective days. Direct feeding on live hamsters resulted in a rate of 96 and 90% for Ae. aegypti and An. dirus, respectively. Although egg production rates decreased from the day 5 feeding to the day 25 feeding, all of the developmental stages resulting from An. dirus fed at day 5 and 15 showed insignificant differences when compared with direct feeding on the blood of a hamster.     

Bionomics of Aedes aegypti subpopulations (Diptera: Culicidae) from Argentina

Differences in biological features of immature and adult Aedes aegypti, as well as variability in vector competence, seem consistent with the existence of genetic variation among subpopulations and adaptation to local conditions. This work aims to compare the bionomics of four Ae. aegypti subpopulations derived from different geographical regions reared under temperate conditions. Life statistics of three Ae. aegypti subpopulations from the provinces of Córdoba, Salta, and Misiones were studied based on horizontal life tables. The Rockefeller strain was used as a control. The development time required to complete the larva and pupa stages varied from 6.91 to 7.95 and 1.87 to 2.41 days, respectively. Significant differences were found in mean larval development time between the Córdoba and Orán subpopulations. The larva‐pupa development time was similar in all the subpopulations. However, survival values varied significantly between the Orán and San Javier subpopulations. The proportion of emergent males did not differ from females within each subpopulation nor among them. Adult longevity was similar among the subpopulations. The average number of eggs laid by each female was significantly different. The Rockefeller strain laid a significantly greater number of eggs (463.99 eggs/female) than the rest of the subpopulations. Moreover, differences in the demographic growth parameter R_o were detected among the four subpopulations. The differences obtained in larval development time, larva‐pupa survival values, and net reproductive rates among the subpopulations might reflect underlying genetic differences as a result of colonization from different regions that probably involve adaptations to local conditions.     

Insecticide susceptibility status of Aedes aegypti (Diptera: Culicidae) from Townsville

Insecticide susceptibility of a 1995 Townsville Aedes aegypti (L.) population was investigated in the laboratory according to World Health Organization guidelines. Using baseline data from two Townsville populations (1955 and 1989), larval bioassays detected significant increases in susceptibility to synthetic pyrethroids and malathion, and significant reductions in susceptibility to most organophosphates and propoxur. Adult bioassays detected significant resistance to bendiocarb and DDT. Comparison of larval data with the international reference ROCK strain showed substantial resistance to have developed to malathion and fenthion. Further analysis revealed the presence of distinct substrains in the baseline 1989 population, which displayed varying levels of temephos susceptibility. We concluded that susceptibility investigations should assess mosquito populations collected from many sites within an area rather than taking a single population from one site, and that the 1989 Ae. aegypti colony would be unsuitable for use as a susceptible reference population.     

Field optimisation of MosquiTRAP sampling for monitoring Aedes aegypti Linnaeus (Diptera: Culicidae)

A sticky trap designed to capture gravid Aedes (Stegomyia) aegypti mosquitoes, MosquiTRAP, has been evaluated for monitoring this species in Brazil. However, the effects of trap densities on the capture rate of Ae. aegypti females and the sensitivity of vector detection are still unknown. After a preliminary study has identified areas of high and low female mosquito abundance, a set of experiments was conducted in four neighbourhoods of Belo Horizonte (state of Minas Gerais, Brazil) using densities of 1, 2, 4, 8, 16, 32 and 64 traps per block. Trap sensitivity (positive MosquiTRAP index) increased significantly when 1-8 MosquiTRAPs were installed per block in both high and low abundance areas. A strong fit was obtained for the total number of mosquitoes captured with increasing trap densities through a non-linear function (Box-Lucas) (r2 = 0,994), which likely exhibits saturation towards an equilibrium level. The capacity of the Mean Female Aedes Index to distinguish between areas of high and low Ae. aegypti abundance was also investigated; the achieved differentiation was shown to be dependent on the MosquiTRAP density.     

The chemical characterization of the epicuticular hydrocarbons of Aedes aegypti (Diptera: Culicidae)

The chemical characterization of the hydrocarbon fraction of the epicuticular lipids of the vector mosquito Aedes aegypti (Linnaeus) was performed using gas chromatography (GC) and gas chromatography-electron impact mass spectrometry (GC-MS). Seventy eight compounds were detected in purified hexane extracts and of these, 42 hydrocarbons were identified and several of the remaining compounds were partially characterized. The hydrocarbon classes present were n-alkanes, monomethylalkanes, dimethylalkanes and alkenes and the results were similar to those published for other Aedes species. Quantitative comparisons of cuticular hydrocarbon profiles were made between males and females, different age groups and between a standard laboratory strain and a recently colonized strain of A. aegypti. These results provide baseline data for further studies on the possible role of mosquito cuticular hydrocarbons in the modification of mating behaviour.     

Field evaluation of effectiveness of the BG-Sentinel, a new trap for capturing adult Aedes aegypti (Diptera: Culicidae)

In recent years, the development of new tools to gather field information about vector ecological parameters has increased. This report evaluated the BG-Sentinel Trap (BGS-Trap), a promising new attempt to improve collection of the dengue vector, Aedes aegypti. The efficacy of the BGS-Trap was compared with the CDC backpack aspirator, one of the commonest used methods for capturing adult mosquitoes. BGS-Traps captured significantly more Ae. aegypti males (chi2 = 21.774, df = 1, P < 0.05) and females (chi2 = 56.007, df = 1, P < 0.05) than CDC aspirator during all days of field collection. However, CDC aspirator was significantly more efficient to capture Culex quinquefasciatus males (chi2 = 5.681, df = 1, P < 0.05) and females (chi2 = 6.553, df = 1, P < 0.05). BGS-Traps captured host-seeking females (varying between 68.75 to 89.8%) in detriment of females in other behavioral and physiological stages. BGS-Traps proved to be efficient and can be used for monitoring adult mosquito populations.     

Pathogenicity of entomopathogenic fungus,Metarhizium anisopliae MET-GRA4 isolate on dengue vectors,Aedes albopictus and Aedes aegypti mosquito larvae (Diptera: Culicidae)

Considering the rapid transmission of the dengue virus, substantial efforts need to be conducted to ward-off the epidemics of dengue viruses. The control effort is depending on chemical insecticides and had aroused undesirable conflicts of insecticide resistance. Here, we study the entomopathogenic fungus, Metarhizium anisopliae as a promising new biological control agent for vector control. The pathogenicity effects of Metarhizium anisopliae against field and laboratory strains of Aedes albopictus and Aedes aegypti larvae were tested using the larvicidal bioassay technique. The results demonstrate that the treatments using M. anisopliae isolate MET-GRA4 were highly effective and able to kill 100% of both Ae. albopictus and Ae. aegypti mosquito larvae at a conidia concentration of 1 × 10?/ml within 7 days of the treatment period. The fungus displayed high larvicidal activity against laboratory and field strain of Ae. aegypti larvae with LC₅₀ values (9.6 × 10³/ml, 1.3 × 10³/ml) and LC₉₅ values (1.2 × 10?/ml, 5.5 × 10⁵/ml) respectively. For Ae. albopictus, LC₅₀ values for laboratory and field strains were (1.7 × 10⁴/ml, 2.7 × 10⁴/ml) and the LC₉₅ values were (2.1 × 10?/ml, 7.0 × 10⁵/ml) respectively. Interestingly, the susceptibility of field strain towards M. anisopliae was higher as compared to the laboratory strain Aedes larvae. In which, the causative agents of all the dead larvae were verified by the virulence of M. anisopliae and caused morphological deformities on larval body. The findings from this study identify this isolate could be an effective potential biocontrol agent for vector mosquitoes in Malaysia.     

Natural infection of the tree hole breeding mosquito Aedes triseriatus (Diptera: Culicidae) with the fungus Funicularius triseriatus

In the course of a study of the population of Aedes triseriatus, a female adults was found to be infected with a fungus. The fungus, Funicularius triseriatus, is described as a new genus and species of the class Hyphomycetes (form-class Deuteromycetes). F. triseriatus is characterized by thallic arthrospores produced on conidiophores arising from hyphae which are funiculose in habit. This is the first report of the natural occurrence of a fungus on A. triseriatus adults.     

The effect of sugar meals and body size on fecundity and longevity of female Aedes communis(Diptera: Culicidae)

Abstract. Starved females of Aedes communis (DeGeer) had significantly shorter mean lifespans than females with access to one blood meal and/or sugar meals. For the starving females, lifespan was significantly correlated with wing length. This correlation was not found in sugar- and/or blood-fed females. Fructose and sucrose had similar effects on mosquito lifespan. The 25% and 50% (w/w) solutions of each, increased mean lifespans to 37.8 ± 7.2 and 34.9 ± 9.1 days, respectively. A 10% solution, of either sugar, was significantly inferior, mean lifespan 23.2 ± 10.2 days. The initial sugar meal was very important for female longevity. Subsequent meals aided in ovarian development. One sugar meal, one blood meal, and several sugar meals during oogenesis was found to be the optimal diet for the female mosquitoes in this study.     

Long-lasting effects of a Bacillus thuringiensis serovar israelensis experimental tablet formulation for Aedes aegypti (Diptera: Culicidae) control

Dengue is a growing public health problem in many tropical and subtropical countries worldwide. At present, the only method of controlling or preventing the disease is to eliminate its vector, Aedes aegypti (L.) (Diptera: Culicidae). In the current study, an experimental larvicide tablet formulation XL-47 based on Bacillus thuringiensis serovar israelensis (Bti) and containing 4.8% of technical powder was developed. This formulation was evaluated against Ae. aegypti in three different sets of experiments, under field-simulated conditions: two experiments were indoors and under partial sunlight exposure and one experiment was outdoors with sunlight exposure. Larvae were added throughout the experiment two times per week, and the residual larvicidal activity was recorded daily. Pupal formation was reduced in the containers with Bti by > 80% in relation to the containers without treatment for 12 wk; to our knowledge, this is the longest period of control reported for a Bti tablet formulation outdoors under sunlight exposure. Moreover, samples from the top, middle, and bottom of the water column were collected to perform bacterial plate counts and toxicity assays. The Bti population and the active ingredient of the tablet formulation remained mainly at the bottom of the containers and mosquito larvae reached the formulation by diving and shredding the tablet's material. In conclusion, the experimental tablet formulation XL-47 showed an inhibition of pupal formation that lasted for long periods under sunlight exposure.     

Diurnal test periods influence behavioral responses of Aedes aegypti (Diptera: Culicidae) to repellents

There is a broad understanding of the influence of environmental factors on various aspects of normal mosquito behavior. How these external factors influence responses to repellent compounds is far less clear. The objective of this study was to investigate the effect of different daytime periods combining the normal circadian activity of a laboratory colony of Aedes aegypti (L.) with behavioral responses of mosquitoes exposed to three different compounds possessing repellent properties. Using an excito-repellency test chamber with different test designs (contact irritancy + repellency and noncontact repellency), female mosquitoes were exposed to each chemical or matching blank control during four different 3-h time intervals beginning 0600 to 1800?h. Mosquitoes showed more significant avoidance responses (escape movement away from the chemical) when exposed to either DEET or hairy basil during the afternoon periods. With deltamethrin, there was no significant difference in repellent escape movement during any period of testing. Escape activity with deltamethrin was significantly greater during all diurnal periods in contact tests compared to DEET and hairy basil. From this study, it was shown that time of diurnal testing can significantly influence behavioral responses of Ae. aegypti exposed to chemical-based repellents. Therefore, the assessment of chemicals (toxins, repellents, attractants) and must carefully consider time-of-test as a potential confounding factor during evaluation and comparisons.     

Characterization of a leucokinin binding protein in Aedes aegypti (Diptera: Culicidae) Malpighian tubule

The insect myokinin (leucokinin-like) neuropeptide family includes peptides that have different physiological effects such as the induction of hindgut myotropic activity and stimulation of urine production. The C-terminal pentamer of myokinins Phe-X-(Ser/Pro/Ala)-Trp-Gly-amide [X=Phe, His, Asn, Ser or Tyr], had been previously determined as the minimum fragment able to elicit a functional response. The receptor(s) for these insect neuropeptides has not yet been identified. In order to characterize the Malpighian tubule leucokinin-like peptide receptor(s) from the yellow fever mosquito (Aedes aegypti), a leucokinin photoaffinity analogue (LPA) of sequence dAla-dTyr-Bpa-dLys-Phe-Phe-Ser-Trp-Gly-amide was designed based on structure/activity relationships for leucokinins. LPA caused depolarization of the transepithelial voltage (TEV) in female Malpighian tubule, confirming the activity of the peptide. The effective concentration to give half the maximum depolarization (EC₅₀) was 17 nM. The ¹²⁵I-LPA was then used to characterize leucokinin binding proteins in female Malpighian tubule membranes. It specifically labeled and saturated a protein(s) of about 54 kDa as shown by SDS-PAGE/autoradiography and by competition experiments with excess unlabeled leucokinin analogues. ¹²⁵I-LPA bound to the 54 kDa protein(s) with a K_d value of 13±3 nM in agreement with the EC₅₀ for the TEV bioassay. Altogether these data suggest that the 54 kDa protein is an Aedes-leucokinin receptor. This is the first characterization of an insect leucokinin receptor and reveals that LPA is a powerful tool to label insect myokinin receptors.     

Evaluation of Mesocyclops annulatus (Copepoda: Cyclopoidea) as a control agent of Aedes aegypti (Diptera: Culicidae) in Argentina

We evaluated the potential of Mesocyclops annulatus as a control agent of Aedes aegypti in La Plata city (Argentina). Mosquito larval survivorship due to predation by these copepods was estimated at weekly intervals during the oviposition period of A. aegypti. Mean weekly A. aegypti larval survivorship in cylindrical plastic containers (12 cm height and 11 cm diameter) with copepods was significantly lower than in control containers. Furthermore, weekly larval survival was negatively correlated with M. annulatus adult density, and approximately 23 adult copepods/container would be a threshold density over which the weekly mosquito larval survivorship approached zero. The copepods were able to persist in all containers during approximately 100 days (in three of them until the end of the experiment: 155 days) without the resource represented by A. aegypti larvae. The predation and persistence observed suggest that M. annulatus is a potential control agent to be considered in biological control programs.