Lipid metabolism during sequential gonotrophic cycles in large and small female Aedes aegypti

The lipid metabolism was investigated during six gonotrophic cycles of Aedes aegypti. Females of constant body size were analyzed for their total lipid content: large females with a body size of 41.06 (wing length cubed) and small females with 15.63. Their lipid contents at eclosion were compared to lipid values after two days of sugar-feeding, shortly before a blood meal, after oviposition, of their total egg batches, and again before the next blood meal, with intermittent access to sugar for two days for six gonotrophic cycles.Large females transferred most of their pre-blood meal lipid into the ovaries. Their low lipid content after oviposition was restored by synthesis from intermittent sugar meals. After the third gonotrophic cycle, they withheld more and more of the resynthesized lipid in their fat body, thus gradually reducing their fecundity. Since blood consumption was not altered significantly during these six cycles, age-related reduction of fecundity was clearly caused by limitations of yolk lipid.Small females transferred a considerably smaller, but constant segment of sugar-derived lipids to the ovaries. In both size classes, lipid content per oocyte was constant throughout all cycles with 9 mcal/oocyte in large and 7 mcal/oocyte in small females. Total fecundity reached 450 eggs in large and 280 eggs in small females. Large females that were maintained on water without sucrose took large blood meals from which part of the yolk lipid was synthesized. Extrapolations suggest that only one or two additional gonotrophic cycles would be possible without additional carbohydrate sources, despite lipogenesis from blood protein.     

Utilization of pre-existing energy stores of female Aedes aegypti mosquitoes during the first gonotrophic cycle

Pre-existing energy reserves may play an important role in regulating the utilization of blood meal proteins in female anautogenous mosquitoes. Determining the fate of reserves derived from the sugar meal and larval food during the first gonotrophic cycle would help to elucidate the relative contributions of larval and adult nutrition to survival and reproduction. We measured the allocation of pre-blood-meal reserves to egg production or energy production during the first gonotrophic cycle by using [14C]-labeled female Aedes aegypti mosquitoes. Feeding adults [3,4-14C]-glucose labeled the glycogen and sugar stores (approximately 50%), lipid stores (approximately 25%), and protein and amino acid stores (approximately 25%). During the first gonotrophic cycle, about 60% of the glycogen and sugar stores were metabolized and all were used for energy production. About 33% of the labeled protein and 72% of the labeled amino acid stores were metabolized, with about 9% being transferred to the eggs and the rest oxidized. About 30% of the lipid was metabolized, with about 65% being transferred to the eggs and the rest oxidized. Feeding [1-14C]-oleic acid to larvae effectively labeled adult lipid stores with about 75% of the label in lipid stores and 16% in proteins and 6% in glycogen. During the first gonotrophic cycle, about 35% of the labeled lipid stores were metabolized, with equal amounts being oxidized and transferred to the eggs. None of the other maternal stores labeled by fatty acid were metabolized during the first gonotrophic cycle. These results show that carbohydrate reserves are a critical source of energy during the first gonotrophic cycle, while lipid reserves are used equally for energy production and provisioning the eggs.     

The programming of circadian flight-activity in relation to mating and the gonotrophic cycle in the mosquito, Aedes aegypti

ABSTRACT. The circadian flight activity of female Aedes aegypti (L.) changes after insemination and during the course of the gonotrophic cycle. Virgin females attain a high level of spontaneous activity within 3–4 days of adult emergence; in LD 12:12 there are peaks of activity at the beginning and end of the light phase. Inseminated females are less than 20% as active as virgins, but exhibit a similar daily pattern. After blood-feeding, inseminated females become almost totally inactive for about 48h and then show a unimodal pattern of activity which is consistent with oviposition behaviour; after oviposition they revert to the basic inseminated pattern and level of activity. Blood-fed virgin females continue to show a high level of spontaneous activity. It is suggested that insemination has the general effect of raising the threshold for activity and that it is necessary before blood-fed females can switch to the gravid behaviour programme. Blood-fed uninseminated females thus continue with behaviour which will maximize the probability of insemination.     

Behavioral and molecular responses of Aedes aegypti to ultrasound

Mosquito-borne infectious diseases cause mortality and global infectious disease burden worldwide. There are several electronic mosquito repellents (EMRs) based on ultrasound have been developed and commercialized to reduce human-mosquito contacts. However, the efficacy of EMRs against mosquitoes is still unclear. In this study, we present experimental evidence that ultrasound of different frequency and sound pressure differentially affects the host-seeking behavior of Aedes aegypti females. Behavioral tests were accompanied by molecular experiments to check whether mosquitoes respond to ultrasound and are there any changes in specific mRNA expression. Experiments in bioassays revealed that the ultrasound of 100 kHz frequency and 90–110 dB pressure significantly disrupted CO?-oriented olfactory behaviors and blocked indoor invasion. Furthermore, a long time (>24 h) exposure to 100 kHz frequency/90 dB pressure of ultrasound decreased attractive behaviors to human skin. At the molecular level, there was no change in expression of odorant receptor co-receptor (AaOrco) in ultrasound treated animals, while one of the CO₂ receptor genes, AaGr3, and putative hearing-related gene, AAEL009258, were down-regulated and up-regulated, respectively. Our study indicates that high frequency (100 kHz) and pressure (90–110 dB) of the ultrasound has repellent effects to olfactory–driven behaviors of mosquitoes.     

Parity and gonotrophic discordance of females of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in the city of São Paulo,SP, Brazil

The objective of this study was to assess the parity, presence of blood in the stomach, and the gonotrophic discordance of females of Aedes aegypti and Aedes albopictus captured in two areas of the city of São Paulo. The captures were undertaken monthly, by aspiration, in the period from January, 2015 to August, 2017. All the females of the two species had their midguts and ovaries dissected to determine the presence of blood and the parity/stage of maturation. With regard to parity, 27% and 34% of the females of Ae. aegypti and Ae. albopictus, respectively, were parous or were in advanced stages of the development of their ovaries (33% and 27%, respectively). The larger part of the females of Ae. aegypti and Ae. albopictus contained blood in their stomachs (77% and 60%, respectively), beyond which 36% and 27% of the females of Ae. aegypti and Ae. albopictus, respectively, were in gonotrophic discordance. Our results indicate favorable conditions in the study areas because of the presence of parous females. Moreover, this frequent and multiple contact of Ae. aegypti and Ae. albopictus females with vertebrate hosts, such as humans, increases the possibility of transmitting the viruses they may be carrying.     

Developing new approaches for detecting and preventing Aedes aegypti population outbreaks: basis for surveillance, alert and control system

A new approach to dengue vector surveillance based on permanent egg-collection using a modified ovitrap and Bacillus thuringiensis israelensis(Bti) was evaluated in different urban landscapes in Recife, Northeast Brazil. From April 2004 to April 2005, 13 egg-collection cycles of four weeks were carried out. Geo-referenced ovitraps containing grass infusion, Bti and three paddles were placed at fixed sampling stations distributed over five selected sites. Continuous egg-collections yielded more than four million eggs laid into 464 sentinel-ovitraps over one year. The overall positive ovitrap index was 98.5% (over 5,616 trap observations). The egg density index ranged from 100 to 2,500 eggs per trap-cycle, indicating a wide spread and high density of Aedes aegypti (Diptera: Culicidae) breeding populations in all sites. Fluctuations in population density over time were observed, particularly a marked increase from January on, or later, according to site. Massive egg-collection carried out at one of the sites prevented such a population outbreak. At intra-site level, egg counts made it possible to identify spots where the vector population is consistently concentrated over the time, pinpointing areas that should be considered high priority for control activities. The results indicate that these could be promising strategies for detecting and preventing Ae. aegypti population outbreaks.     

A preparation method for Aedes aegypti and Aedes atropalpus chorions for protein characterization

This investigation presents a method of preparation and comparison of the fully-formed eggshells (chorions) of the aedine mosquitoes, Aedes aegypti and Aedes atropalpus, by a gentle lysis-washing protocol. Light and scanning electron micrographs of the prepared chorions display a two-layered structure in the characteristically sculptured endochorion and the tubercle-patterned exochorion. SDS-gel electrophoresis of solubilized whole ovaries and prepared chorions, stained for protein and carbohydrate, show significant differences between the two species mainly in the chorion electrophoresis patterns.     

Influence of the spatial distribution of human hosts and large size containers on the dispersal of the mosquito Aedes aegypti within the first gonotrophic cycle

It is generally accepted that Aedes aegypti (L.) (Diptera: Culicidae) has a short dispersal capacity, and that displacement can be influenced by the availability of oviposition sites in the surroundings of emergence or release sites. In the present article, we observed the influence of spatial heterogeneity of large containers and human hosts on the cumulative flight direction of Ae. aegypti females during the first gonotrophic cycle, testing the hypothesis that they aggregate in resource-rich areas, i.e. where there are higher concentrations of large containers and/or humans per habitation. We analysed data from pupal surveys and mark-release-recapture experiments (non-blood-fed females were released) carried out in two dengue endemic neighbourhoods of Rio de Janeiro, Brazil: Tubiacanga (a suburb, with a human density of 337 inhabitants/ha) and Favela do Amorim (a slum, with a human density of 901 inhabitants/ha). In both areas, host-seeking females of three different release cohorts showed an overall non-uniform and extensive dispersal from their release point within 1–2 days post-release. At 4–5 days post-release, when many of the released females would be expected to be gravid, in Tubiacanga most mosquitoes were collected in areas with a relatively higher density of containers/premise, independently of the density of residents/house, whereas in Favela do Amorim, almost half of the captured mosquitoes were collected in relatively resource-poorer areas. Although Ae. aegypti dispersal patterns varied between sites, overall the distances travelled from the release point and the cumulative flight directions were correlated with the density of containers and hosts, more markedly in Tubiacanga than in Favela do Amorim.     

A new tent trap for monitoring the daily activity of Aedes aegypti and Aedes albopictus

In this study, we designed a new tent trap; the BioDiVector (BDV) tent trap, consisting of two rectangular tents that use human bait without endangering the technical personnel. The daily activity pattern of Aedes aegypti and Aedes albopictus in intra, peri, and extradomiciliary sites was studied in an endemic area of dengue in southern Mexico by using the BDV tent trap. Totals of 3,128 individuals of Ae. aegypti and 833 Ae. albopictus were captured. More Ae. aegypti males than females were caught, while the opposite was true with Ae. albopictus. The activity of both mosquito species was affected by the interaction between the collection site and time of day. In general, more individuals of both mosquito species were captured at the extradomicillary sites than at the peri and intradomicillary sites. Mosquitoes showed two peaks of activity, one in the morning and the other in the afternoon, but in general this only occurred at the extradomicillary sites, whereas no peak of activity was observed at the intra and peridomicillary sites. Overall, Ae. aegypti had a higher indirect biting rate than Ae. albopictus. Finally, due to its efficiency, simplicity, and low cost, we suggest the use of this innovative tool for entomological surveillance, bionomics and vector incrimination studies in geographical areas where dengue and other arboviruses are present.     

The Aedes aegypti genome: complexity and organization.

We describe the use of DNA reassociation kinetics to determine the total genome size and complexity together with the individual complexity and copy number of the single copy, middle repetitive and highly repeated DNA fractions of cell line and larval DNA from the mosquito, Aedes aegypti. The genome of Ae. aegypti is both large and complex, being one third the size of the human genome, and exhibits a short period interspersed repeat pattern. The implications of patterns of sequence arrangement and genome complexities for experiments aimed at isolating specific classes of DNA sequences, such as mobile genetic elements, are discussed.     

Different Repellents for Aedes aegypti against Blood-Feeding and Oviposition

Methyl N,N-dimethyl anthranilate (MDA), ethyl anthranilate (EA) and butyl anthranilate (BA) were previously shown to repel Aedes aegypti mosquitoes from landing on human skin. However, the effect of these compounds on the orientation of flying mosquitoes in a choice situation and their effect on mosquito oviposition are not yet known. Here, we used a modified Y-tube olfactometer to test the effect of these compounds on the orientation of Aedes aegypti flying towards skin odor (human fingers), and we tested their effect on Aedes aegypti oviposition choice in a cage assay. In both behavioral situations we compared the effect to the well-documented repellent N,N-diethyl-meta-toluamide (DEET). MDA, EA, and DEET inhibited Aedes aegypti from flying towards skin odor while BA had no such effect. Conversely, MDA had no effect on oviposition while EA, BA, and DEET deterred oviposition, with the strongest effect observed for BA. Thus, we confirm that EA and DEET are generally repellent, while MDA is repellent only in a host-seeking context, and BA is deterrent only in an oviposition context. These compounds appear of potential use in mosquito control programs.     

A secure semi-field system for the study of Aedes aegypti

Background

New contained semi-field cages are being developed and used to test novel vector control strategies of dengue and malaria vectors. We herein describe a new Quarantine Insectary Level-2 (QIC-2) laboratory and field cages (James Cook University Mosquito Research Facility Semi-Field System; MRF SFS) that are being used to measure the impact of the endosymbiont Wolbachia pipientis on populations of Aedes aegypti in Cairns Australia.

Methodology/Principal Findings

The MRF consists of a single QIC-2 laboratory/insectary that connects through a central corridor to two identical QIC-2 semi-field cages. The semi-field cages are constructed of two layers of 0.25 mm stainless steel wire mesh to prevent escape of mosquitoes and ingress of other insects. The cages are covered by an aluminum security mesh to prevent penetration of the cages by branches and other missiles in the advent of a tropical cyclone. Parts of the cage are protected from UV light and rainfall by 90% shade cloth and a vinyl cover. A wooden structure simulating the understory of a Queenslander-style house is also situated at one end of each cage. The remainder of the internal aspect of the cage is covered with mulch and potted plants to emulate a typical yard. An air conditioning system comprised of two external ACs that feed cooled, moistened air into the cage units. The air is released from the central ceiling beam from a long cloth tube that disperses the airflow and also prevents mosquitoes from escaping the cage via the AC system. Sensors located inside and outside the cage monitor ambient temperature and relative humidity, with AC controlled to match ambient conditions. Data loggers set in the cages and outside found a <2°C temperature difference. Additional security features include air curtains over exit doors, sticky traps to monitor for escaping mosquitoes between layers of the mesh, a lockable vestibule leading from the connecting corridor to the cage and from inside to outside of the insectary, and screened (0.25 mm mesh) drains within the insectary and the cage. A set of standard operating procedures (SOP) has been developed to ensure that security is maintained and for enhanced surveillance for escaping mosquitoes on the JCU campus where the MRF is located. A cohort of male and female Aedes aegypti mosquitoes were released in the cage and sampled every 3–4 days to determine daily survival within the cage; log linear regression from BG-sentinel trapping collections produced an estimated daily survival of 0.93 and 0.78 for females and males, respectively.

Conclusions/Significance

The MRF SFS allows us to test novel control strategies within a secure, contained environment. The air-conditioning system maintains conditions within the MRF cages comparable to outside ambient conditions. This cage provides a realistic transitional platform between the laboratory and the field in which to test novel control measures on quarantine level insects.     

Cysteine sulfinic acid decarboxylase activity of Aedes aegypti aspartate 1-decarboxylase: the structural basis of its substrate selectivity

Insect aspartate 1-decarboxylase (ADC) catalyzes the decarboxylation of aspartate to β-alanine. Insect ADC proteins share high sequence identity to mammalian cysteine sulfinic acid decarboxylase (CSADC), but there have been no reports indicating any CSADC activity in insect ADC or any ADC activity in mammalian CSADC. Substrate screening of Aedes aegypti ADC (AeADC), however, demonstrates that other than its activity to aspartate, the mosquito enzyme catalyzes the decarboxylation of cysteine sulfinic acid and cysteic acid as efficiently as those of mammalian CSADC under the same testing conditions. Further analysis of Drosophila melanogaster ADC also demonstrated its CSADC activity, suggesting that all insect ADC likely has CSADC activity. This represents the first identification of CSADC activity of insect ADC. On the other hand, HuCSADC displayed no detectable activity to aspartate. Homology modeling of AeADC and substrate docking suggest that residue Q377, localized at the active site of AeADC, could better interact with aspartate through hydrogen bonding, which may play a role in aspartate selectivity. A leucine residue in mammalian CSADC occupies the same position. A mutation at position 377 from glutamine to leucine in AeADC diminished its decarboxylation activity to aspartate with no major effect on its CSADC activity. Comparison of insect ADC sequences revealed that Q377 is stringently conserved among the available insect ADC sequences. Our data clearly established the CSADC activity of mosquito and Drosophila ADC and revealed the primary role Q377 plays in aspartate selectivity in insect ADC.