Female Aedes Aegypti Research Articles

Synergistic attraction of Aedes aegypti (L.) to binary blends of L-lactic acid and acetone, dichloromethane, or dimethyl disulfide.

Kairomones produced by humans provide female anthropophilic mosquitoes with vital cues used in host-seeking for a blood meal. These chemicals are emanated primarily by the skin and provide the mosquitoes a means to orient themselves to humans at a relatively close range. Chemical studies of these emanations have provided new ideas for the formulation of attractant blends. We report mosquito attraction responses for three binary blends and their separate components. The blends are comprised of L-lactic acid plus either acetone, dichloromethane, or dimethyl disulfide. At the emission rates used in our bioassays, these blends synergistically attract laboratory-reared female Aedes aegypti. Carbon dioxide is not a necessary component to yield high levels of attraction with these blends. It is postulated that at least one of these synergistic blends (L-lactic acid and acetone) produces mosquito attraction behavior similar to L-lactic acid and CO2.

Free amino acids are important for the retention of protein and non-protein meals by the midgut of Aedes aegypti females

There is a relationship between the normal progress of digestion and the retention or elimination of the proteins ingested with the meal by Aedes aegyti females. The addition of soybean trypsin inhibitor (STI) to a protein meal prevented digestion and resulted in a rapid elimination of the undigested proteins. The addition of a mix of free amino acids to a protein meal together with STI resulted in a significant increase in the retention of the undigested proteins during the first 10–15 hrs after feeding. The effect of the free amino acids on the retention of the proteins was concentration-dependent between 250 μg/ml and 5 mg/ml. Free amino acids were also important for the retention of non-protein meals. When females were fed a meal containing FITC-dextran (20 kD), most of this compound was eliminated into the feces by 10 hrs; the addition of free amino acid resulted in a significant increase in the retention of the FITC-dextran by the midgut during the first 15 hrs after feeding. The presence of free amino acids in the midgut lumen seems to be an important signal used by the mosquito to regulate the retention of the meal.

Benzoquinones from millipedes deter mosquitoes and elicit self-anointing in capuchin monkeys (Cebus spp.).

Neotropical monkeys of the genus Cebus anoint themselves by rubbing arthropods and plants against their pelage. A recent study has shown that free-ranging wedge-capped capuchin monkeys (C. olivaceus) in Venezuela self-anoint with a benzoquinone-secreting millipede, an activity by which they are hypothesized to appropriate chemical deterrents of mosquitoes. To evaluate the plausibility of this hypothesis, female yellow fever mosquitoes (Aedes aegypti) were presented with two millipede secretory compounds, 2-methyl-1,4-benzoquinone and 2-methoxy-3-methyl-1,4-benzoquinone, on nylon-reinforced silicone membranes placed over wells filled with human blood, a highly preferred food. Mosquitoes exhibited fewer landings, fed less frequently, and flew more frequently (a possible indication of repellency) in the presence of membranes treated with benzoquinones than with controls. These compounds also elicit self-anointing in captive male and female tufted (C. apella) and white-faced (C. capucinus) capuchin monkeys.

Proline can be utilized as an energy substrate during flight of Aedes aegypti females

In order to determine whether proline can be utilized as fuel during flight of Aedes aegypti, proline, alanine, and glutamine concentrations were monitored at 0, 30 and 60 min after flight using sugar-fed males and females, and blood meal-fed females. In sugar-fed and blood meal-fed females, flight lead to a significant decrease in proline and a significant increase in glutamine concentration in both hemolymph and thorax. Only during flight after a blood meal was a significant increase in the alanine concentration observed in hemolymph. After flight, the proline alanine and glutamine levels in the hemolymph and thorax from males did not change significantly. In addition, activities of enzymes related to amino acid metabolism were assayed in homogenates of cephalothorax and thorax from both sexes, and in fat body and midgut from females. In both sexes, the activities of all the enzymes studied were significantly higher in thorax than in cephalothorax. The levels of the enzymes involved in proline oxidation were higher in thorax than in fat body and midgut. These results suggest that proline can be used as an energy substrate for flight muscle of Ae. aegypti females. However, the elevation in glutamine levels observed in hemolymph and thorax after flight has not been reported in other insects that fuel flight using proline and may suggest an additional mechanism for shuttling ammonia between flight muscle and fat body is present in mosquitoes.

The role of hemolymph proline as a nitrogen sink during blood meal digestion by the Mosquito Aedes aegypti

Mosquitoes utilize the amino acids derived from blood meal protein to produce egg proteins. But the amino acids can also be used to produce egg lipid or can be oxidized for energy production. These latter two processes result in the release of nitrogen as toxic ammonia. Therefore, amino acids must be processed in such a way that amino acid nitrogen can be incorporated into non-toxic waste products. Proline is the predominant amino acid in the hemolymph of the adult female mosquito Aedes aegypti. After feeding on albumin meal, hemolymph proline levels increased five-fold over unfed levels, reached maximal levels in the first hours after feeding and remained high through oviposition. Hemolymph proline levels increased as the concentration of protein in the meal increased. When starved of sugar for 24 h prior to feeding on an albumin meal, hemolymph proline levels increased four-fold over the proline levels of non-starved mosquitoes. Proline levels after feeding on a protein deficient in essential amino acids, pike parvalbumin, increased to twice the levels of albumin fed mosquitoes. Based on these observations, we propose that mosquitoes utilize proline as a temporary nitrogen sink to store ammonia arising from deamination of blood meal amino acid.

DENGUE AND ITS VECTORS IN THAILAND: CALCULATED TRANSMISSION RISK FROM TOTAL PUPAL COUNTS OF AEDES AEGYPTI AND ASSOCIATION OF WING-LENGTH MEASUREMENTS WITH ASPECTS OF THE LARVAL HABITAT

Working in a village dengue focus in Chachoengsao Province, Thailand, aedine mosquito larvae and pupae were counted in all containers of 10 houses per month. The wings of female Aedes aegypti (L.) emerging from pupae were measured. Number of pupae and size of emerging females increased in containers with qualities that favored availability of larval food sources (e.g., uncovered containers). The small size of most mosquitoes compared with those raised in the laboratory indicated that the larval population as a whole was under nutritional stress. Applying the number of pupae per house and measurement of air and water temperature with an existing model, the risk of dengue transmission was greatest in May and June. The estimated number of female Ae. aegypti per house was well above the threshold for increasing transmission in all months but December through February. A phased approach to sampling immature aedine mosquitoes in Thailand is proposed, which would consist of routine surveillance of larval index and occasional total counts with measurement of wing size. Such a system would combine the benefits of the simple application of larval surveillance with the valuable data gathered from pupal counts and wing measurements.

Dengue virus infection rate in field populations of female Aedes aegypti and Aedes albopictus in Singapore.

We developed a single-step reverse transcription-polymerase chain reaction (RT-PCR) followed by a semi-nested PCR using an upstream consensus primer and four type-specific primers within the non-structural protein gene (NS3) of dengue viruses to type dengue viruses in field populations of female Aedes mosquitoes. This yielded diagnostic fragments of 169, 362, 265 and 426 base pairs for dengue virus types 1, 2, 3 and 4, respectively. From 1997 to 2000, 54 (6.9%) of 781 Aedes aegypti and 67 (2.9%) of 2256 Aedes albopictus screened were positive for dengue viruses, with a declining trend. The most common dengue virus type detected in the Aedes mosquitoes was dengue-1. Details on the change of one serotype to another in the mosquito population over three consecutive years are discussed.

Midgut exopeptidase activities in Aedes aegypti are induced by blood feeding

Midgut extracts from Aedes aegypti females exhibited hydrolytic activities against synthetic substrates for carboxypeptidase A, carboxyopeptidase B and leucine-aminopeptidase. The three activities showed a broad pH optimum, with maximum activities at pH between 6.5 and 8.5. Enzymatic activities were further characterized by testing the effects of a variety of protease inhibitors. Captopril and 1-10-phenantroline inhibited the activities of carboxypeptidases A and B, while leuhistin, amastatin and bestatin inhibited aminopeptidase activity. Exopeptidase activities were induced by a blood meal and the highest activities were found during the peak of trypsin activity, about 20–24h after feeding. An amino acid meal failed to induce significant increases in any of the three exopeptidase activities. The amounts of exopeptidase activities induced were proportional to the protein concentration of the meal. The addition of soy-trypsin inhibitor to the protein meal blocked the post-feeding induction of exopeptidases. The features of the induction of synthesis of the three exopeptidase activities resembled the induction of synthesis of late trypsin during the second phase of digestion.

Indirect evidence against delayed "skip-oviposition" behavior by Aedes aegypti (Diptera: Culicidae) in Thailand.

Mosquitoes that do not oviposit their entire batch of eggs in one location, but choose instead to oviposit a few eggs in several different sites exhibit skip oviposition. Although skip oviposition may ensure the greater distribution of progeny from an individual female and reduce sibling competition, it also uses greater maternal energy reserves, may increase the risk of adult female mortality and does not reduce other competition. To test the hypothesis that female Aedes aegypti (L.) do not oviposit all of their eggs at one time and place, we examined females collected with an aspirator resting inside houses in Thailand. Females (n = 384) were dissected and the stage and number of developing oocytes were enumerated. If skip oviposition is a common behavior among female Ae. aegypti, one would expect to find significantly fewer mature eggs (Christophers' stage V) in gravid females than developing oocytes in half-gravid females (Christophers' stage IIIa-IVb). No significant differences were found between ovaries containing immature or mature oocytes, even when the data were stratified by female body size and when immature oocytes that potentially could undergo resorption (Christophers' stages IIIa-IIIb) were excluded from the analysis. We found no evidence that skip oviposition over several days is a common behavior among domestic populations of Ae. aegypti at the end of the rainy season in this region of Thailand.

Why do female Aedes aegypti (Diptera: Culicidae) feed preferentially and frequently on human blood?

Adult female Aedes aegypti (L.), the vector of dengue and yellow fever viruses, have an affinity for feeding on human blood and a tendency to forego feeding on sugar. This observation challenges two tenets of mosquito biology: (1) mosquitoes imbibe plant carbohydrates for synthesis of energy reserves and blood for reproduction and (2) egg production is reduced when mosquitoes feed on human blood compared with blood from other species. Sub-optimal amounts of the amino acid isoleucine in human blood (particularly free isoleucine in plasma) are thought to be responsible for lowered egg production when human blood is ingested. We tested the hypothesis that feeding on human blood is associated with a selective advantage for Ae. aegypti and is an underlying reason for this mosquito's intimate and epidemiologically important relationship with human beings. Our five experiments examined the effects of different isoleucine concentrations on accumulated energy reserves, frequency of host contact, survival, and egg production. When mosquitoes imbibed blood meals over a 7- to 10-d period and were not fed sugar, increased isoleucine concentration decreased energy reserves and did not increase egg production. Aedes aegypti took smaller but more frequent blood meals when feeding on a low-isoleucine human host daily compared with a high-isoleucine mouse host. Previous reports that isoleucine enhances egg production were confirmed only when females were fed sugar, an unusual behavior for most domestic Ae. aegypti populations. Females fed human blood and water had greater age-specific survival (l(x)), reproductive output (m(x)), and cumulative net replacement (R0) than cohorts fed human blood plus sugar or isoleucine-rich mouse blood with or without access to sugar. The unique isoleucine concentration of human blood is associated with Ae. aegypti's unusual propensity to feed preferentially and frequently on humans--a behavior that increases this mosquito's fitness, synthesis of energy reserves, and contact with human hosts, making it an especially effective disseminator of human pathogens.

First isolation of dengue 1 virus from Aedes aegypti in Federal District, Brazil.

Dengue is present in the Federal District since 1991 and virological studies of the vector began in 1998. Two strains of DEN1 were isolated from 9 pools of female Aedes aegypti (78 mosq.), collected in April in Gama county, where the Breteau index was 5.4, and 32 autochtonous human cases were notified.

Aedes aegypti (Diptera: Culicidae) age determination by cuticular hydrocarbon analysis of female legs.

We previously described methods for age-grading female Aedes aegypti (L.) by gas chromatographic (GC) analysis of whole-body cuticular hydrocarbon patterns. Two regression models were developed that were based on the age-dependent, relative abundance of 2 cuticular hydrocarbons, pentacosane (GC peak 1) and nonacosane (GC peak 5). We have refined this method so that only the legs are required to age individual females. Two new regression models were developed that also use the relative abundance of a 3rd cuticular hydrocarbon, octacosane (GC peak 4). These models improve the overall accuracy of the cuticular hydrocarbon method for aging female mosquitoes, especially for older females from 132 to 165 degree-days (DD) of age (12-15 calendar days at 28 degrees C). The correlation coefficients (R2) for the best-fitted linear regression models for aging females from 0 to 132 and 0-165 DD were 0.80 and 0.81, respectively (P < 0.001 in all cases). The use of leg cuticular hydrocarbons for estimating the age of female Ae. aegypti has a significant advantage over whole-body extracts as indicated by the decreased variability associated with the relative abundance of pentacosane and the expanded range over which the models were able to predict age accurately by the addition of the relative abundance of octacosane.

Potential for aging female Aedes aegypti (Diptera: Culicidae) by gas chromatographic analysis of cuticular hydrocarbons, including a field evaluation.

Gas chromatography with flame-ionization detection was used to measure the time-associated, quantitative changes in the cuticular hydrocarbons of female Aedes aegypti (L.). Cohorts of unstressed Ae. aegypti, Rockefeller strain, were reared and held at 3 constant temperatures (24, 28, and 30 degrees C). Five females from each cohort were taken at 33 degree-day (DD) intervals from 0 to 231 DD (using 17 degrees C as the threshold temperature). Quantitative changes over time of cuticular hydrocarbons associated with gas chromatographic peaks 1 and 5 were identified as having promise for age grading. The relative abundance of peak 1 (pentacosane) decreased linearly from 0 to 132 DD, whereas peak 5 (nonacosane) increased linearly over the same period. Suboptimal larval conditions (crowded and starved), which resulted in physiological stress (decreased size), had negligible effect on the relative abundance of pentacosane and nonacosane. Additionally, the rate of change in the relative abundance of pentacosane and nonacosane were the same for both a recently colonized Chachoengsao (Thailand) strain of Ae. aegypti compared with the long-colonized Rockefeller (Caribbean) strain over a 0-99 DD interval. Two linear regression models, one based on the relative abundance of pentacosane and the other on the logit transformation of these values, were developed for aging female Ae. aegypti. A blind study using laboratory-reared mosquitoes and a mark-release-recapture experiment using field mosquitoes validated these age-grading models and produced promising results for aging females up to 132 DD (19, 12, and 10 calendar days at 24, 28 and 30 degrees C, respectively). Therefore the regression models, based on the relative abundance of these 2 cuticular hydrocarbons, appeared to be a useful approach for age-grading Ae. aegypti up to at least 12 d of age regardless of environmental conditions (temperature and stress) and population history (origin and colonization time).

Mosquito hearing: sound-induced antennal vibrations in male and femaleAedes aegypti

Male mosquitoes are attracted by the flight sounds of conspecific females. In males only, the antennal flagellum bears a large number of long hairs and is therefore said to be plumose. As early as 1855, it was proposed that this remarkable antennal anatomy served as a sound-receiving structure. In the present study, the sound-induced vibrations of the antennal flagellum in male and female Aedes aegypti were compared, and the functional significance of the flagellar hairs for audition was examined. In both males and females, the antennae are resonantly tuned mechanical systems that move as simple forced damped harmonic oscillators when acoustically stimulated. The best frequency of the female antenna is around 230 Hz; that of the male is around 380 Hz, which corresponds approximately to the fundamental frequency of female flight sounds. The antennal hairs of males are resonantly tuned to frequencies between approximately 2600 and 3100 Hz and are therefore stiffly coupled to, and move together with, the flagellar shaft when stimulated at biologically relevant frequencies around 380 Hz. Because of this stiff coupling, forces acting on the hairs can be transmitted to the shaft and thus to the auditory sensory organ at the base of the flagellum, a process that is proposed to improve acoustic sensitivity. Indeed, the mechanical sensitivity of the male antenna not only exceeds the sensitivity of the female antenna but also those of all other arthropod movement receivers studied so far.

A new Y‐tube olfactometer for mosquitoes to measure the attractiveness of host odours

AbstractIn a new bioassay a small Y‐shaped wind tunnel is used to quantitatively investigate the responses of mosquitoes to host odours. Female yellow fever mosquitoes, Aedes aegypti (L.) (Diptera: Culicidae) were tested to (1) a human hand, (2) an extract with human skin residues, (3) L‐(+)‐lactic acid, and (4) carbon dioxide. The responses to the skin extract followed a sigmoid dose response curve. The most effective dose attracted 80–90% of the mosquitoes within 30 s and was as effective as the human hand. L‐(+)‐lactic acid was identified in this extract and found attractive for mosquitoes also when presented alone. Carbon dioxide stimulated taking flight and was attractive, an effect which was synergistically enhanced in combination with L‐(+)‐lactic acid. The presented bioassay is especially suited to test the behavioural effects of synthetic odours as well as of natural odour sources. Due to the fast response of the mosquitoes, the sensitivity, and the simplicity of the testing procedure it is a potent tool in the search for new attractive components.

A molecular view of trypsin synthesis in the midgut of Aedes aegypti

Ingestion of a blood meal induces two phases of trypsin synthesis in the midgut of Aedes aegypti females. The first phase, which encompasses the first 4–6 hours following a blood meal, is characterized by the presence of small amounts of early trypsin. The second phase, which occurs between 8 and 36 hours after blood feeding, is characterized by the presence of large amounts of late trypsin. A specific form of regulation of trypsin synthesis characterizes each phase: early trypsin synthesis is regulated at the translational level, while late trypsin synthesis is regulated at the transcriptional level. The enzymatic activity of early trypsin plays a unique and critical role in the regulation of late trypsin synthesis. Early trypsin acts like a “sensor”. It carries out limited proteolysis of the ingested proteins and, somehow, the products of this limited proteolysis induce synthesis of late trypsin, which is the protease responsible for the majority of the endoproteolytic cleavage of the meal proteins. Transcription of the early trypsin gene starts a few hours after adult emergence and is under control of juvenile hormone. However, the early trypsin mRNA is stored in the midgut epithelium and remains untranslated until a blood meal is taken. The exact mechanism responsible for initiating translation is presently unknown, but an increase in the size of the amino acid pool in the midgut is sufficient to activate translation of early trypsin mRNA. The transcription of the late trypsin gene is regulated by uncharacterized proteolysis products generated by the action of early trypsin on the blood meal proteins. Once transcription has been activated, the rate of transcription of the late trypsin gene is proportional to the amount of protein present in the meal. In addition, the amount of late trypsin protein translation is controlled by the amount of amino acid released during digestion. Regulation at both transcriptional and translational levels allows the midgut to adjust the amount of late trypsin with remarkable flexibility in response to a particular meal.

Partitioning of glycogen, lipid, and sugar in ovaries and body remnants of female Aedes aegypti (Diptera: Culicidae) fed human blood.

We examined the accumulation of glycogen, lipid, and sugar obtained from a human blood meal for egg development and body energy reserves by small and large female Aedes aegypti (L.). Small and large mosquitoes were fed a single meal of human blood on day 2 after emergence. Mosquitoes were collected at 4, 12, 24, 48, and 72 h after blood feeding and ovaries and body remnants were separated by dissection and then assayed. Large mosquitoes had greater reserves than small mosquitoes. Mosquitoes deposited similar proportions of lipid reserves obtained from blood meals in their ovaries regardless of body size. Small mosquitoes deposited a significantly higher proportion of their glycogen in ovaries than large mosquitoes. The pattern of energy accumulation and use indicates that to avoid starvation, mosquitoes fed a single human blood meal will need to feed again before ovipositing, and that multiple feeding may be more important for small than for large Ae. aegypti.

Utilization of human blood and sugar as nutrients by female Aedes aegypti (Diptera: Culicidae).

We examined the utilization of sugar and human blood as nutrient sources for small and large female Aedes aegypti (L.) when they were fed blood 2 or 5 d after emergence. Laboratory-reared mosquitoes were fed human blood alone or sugar plus human blood and assayed at 4, 12, 24, and 48 h after the blood meal. Starved and well-fed mosquitoes were obtained by holding teneral females (< or = 1 d old) with 0, 5, 10, and 15% sucrose solutions ad libidum from emergence. Both small and large mosquitoes increased their glycogen and sugar levels significantly by feeding on blood only or on blood plus sugar when they imbibed a human blood meal on day 2 after emergence. Mosquitoes only fed blood on day 2 had the highest lipid levels of any treatment group. Both size classes and all feeding regimes failed to increase the total amount of glycogen, lipid, or sugar when they fed on blood 5 d after emergence. We conclude that there is an energetic advantage to Ae. aegypti when they feed on blood early in adult life (< or = day 2 after emergence).

Mediation of Deet Repellency in Mosquitoes (Diptera: Culicidae) by Species, Age, and Parity

Laboratory bioassays assessed differences in the protection time provided by the repellent deet (N,N-diethyl-3-methylbenzamide) against 5-d-old nulliparous and 10-, 15-, and 20-d-old nulliparous and parous female Aedes aegypti (L.), Anopheles albimanus (Weidemann), and Anopheles quadrimaculatus Say sensu lato. Mean protection time was shortest against An. albimanus (1.6 h) and An. quadrimaculatus (1.5 h) and longest against Ae. aegypti (6.5 h), but was not significantly influenced by mosquito age or parity. Mean percentage of biting at repellent failure time was highest in An. albimanus (14.2%), followed by An. quadrimaculatus (7.0%) and Ae. aegypti (2.9%), was higher in parous females (10.8%) than in nulliparous females (5.9%), and was highest overall (35%) in 20-d-old parous An. albimanus. Interaction between mosquito species and parity and between parity and age factors, respectively, resulted from a significant decrease in percentage of biting by parous An. quadrimaculatus compared with other females, and a significant increase in biting by 20-d-old parous females compared with other females. The main finding of this study is that repellent protection time is unaffected by parity; this is important because parous mosquitoes are the primary target of personal-protection measures in disease-endemic areas. When repellent failure did occur, there was a higher risk of bite by old, parous An. albimanus than for any other species, age, or parity grouping of females.

Influence of plant abundance on nectar feeding by Aedes aegypti (Diptera: Culicidae) in southern Mexico.

The availability of flowering plants affected the sugar feeding rates of female Aedes aegypti (L.) in 4 areas of a small city in southern Mexico. The proportion of mosquitoes containing sugar varied from 8 to 21% in 4 areas in direct relation to blooming plant abundance. Human density was similar in the 4 areas (range, 3.9-5.4 per house), whereas the number of flowering plants per house increased on the outskirts (range, 3.1-5.4 plants per house). Equal proportions of sugar positive females were nulliparous or parous, indicating similar sugar feeding at any age. In addition, nearly 60% of positive females were at the Christophers stage II, indicating a greater need for flight fuel during the early stages of egg development. We conclude that Ae. aegypti feeds frequently on nectar and that this activity is modulated by nectar availability.