A study on the fecundity of male Aedes aegypti

After inseminating about 5 females, the males of Aedes aegypti are sexually depleted and most do not then re-inseminate additional females for the rest of their lives. Sexually depleted males generally replenish spermatozoa in their seminal vesicles and renew the secretion in their accessory glands, i.e. they again become fecund. Many replenished males do not chase or attempt to copulate with virgin females. Those males which do copulate do not ejaculate. Thus, celibate behaviour and failure to ejaculate rather than lack of fecundity are responsible for the inability of sexually depleted males to impregnate females.     

A genetical study of DDT resistance in the mosquito Aedes aegypti.

Crosses have been carried out to determine the relationship between adult DDT resistance and the three linkage groups of the mosquito Aedes aegypti. Two linkage groups were implicated in the control of DDT resistance. In the Bangkok-HR strain resistance derived mainly from linkage group III, probably with the maor effect from the gene R-DDT2. When resistance was transferred into a susceptible background, by outcrossing Bangkok-HR to strain 64 and reselecting, resistance in the resulting Bangkok-MR strain came from both linkage groups II and III.     

Mating compatibility and competitiveness of transgenic and wild type Aedes aegypti (L.) under contained semi-field conditions

We conducted the world??s first experiments under semi-field conditions (ACL-2 field house) to assess the mating competitiveness of genetically sterile RIDL male mosquitoes (513A strain). The field house is a state-of-the-art, fully-contained trial facility, simulating the living space for a household of 2?C4 people in Peninsular Malaysia. Ten genetically sterile RIDL male A. aegypti mosquitoes competed with ten wild type males inside this field house to mate with ten wild type females. Hatched larvae from mated females were screened under a fluorescent microscope for genetic markers to determine if they were fathered by RIDL male or wild type male, and all results were cross-checked by PCR. Two such experiments were conducted, each repeated sufficient number of times. All strains were on a Malaysian lab strain background for the first experiment, while the RIDL males alone were on a recently-colonised Mexican strain background for the second experiment. A total of 52 % of the matings were with RIDL males in the first experiment, while 45 % of the matings were with RIDL (Mexican) males in the second experiment. Statistically, this is not significantly different from 50 % of the matings expected to take place with RIDL males if the latter were as competitive as that of the wild type males. This shows that A. aegypti RIDL-513A has excellent mating competitiveness under semi-field conditions, verifying earlier trends obtained in small lab cages. We also observed high mating compatibility between recently-colonised Mexican RIDL males and lab-reared Malaysian wild type females.     

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.     

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.     

A novel multiple membrane blood‐feeding system for investigating and maintaining Aedes aegypti and Aedes albopictus mosquitoes

A novel multiple membrane blood‐feeding system for mosquitoes has been developed for the study and routine maintenance of Aedes aegypti L. and Aedes albopictus Skuse that require a meal of vertebrate blood to produce eggs. This blood‐feeding system uses cattle collagen sausage‐casing membrane to facilitate feeding. The efficiency of this blood‐feeding system was compared to a live mice blood source. We observed that Ae. aegypti that fed on pig whole blood had 89.7% (w/o ATP) and 90.7% (w/ ATP) blood‐feeding rates, which were not significantly different from the mice‐fed ones (98.0%). Ae. albopictus fed on pig whole blood (w/ ATP) had a success rate of 84.4%, which was significantly different from the mice‐fed mosquitoes (51.1%). The feeding rates did not differ between sausage‐casing membrane and Parafilm‐M®. The survival rate, fecundity, pupation, and pupal emergence rates of Aedes females fed on pig whole blood were not significantly different from the mice‐fed ones. The artificial blood feeder can be applied to replace live animals as blood sources. Considering that this simple, inexpensive, convenient, and efficient feeding device can be built with common laboratory materials for research on Aedes mosquitoes.     

A floral‐derived attractant for Aedes aegypti mosquitoes

The reproductive success of Aedes aegypti (L.) (Diptera: Culicidae) is strongly dependent on the availability of carbohydrates in the environment and the ability of the mosquitoes to locate them. The most significant source of carbohydrates for mosquitoes is nectar from flowering plants, which mosquitoes locate by their volatile compounds. The aim of our work was to identify plant volatile compounds that elicit a behavioral response in Ae. aegypti, which may be included in a mosquito trap for surveillance and/or control purposes. Landing‐preference bioassays were performed with plants of three species—Plectranthus neochilus Schltr. (Lamiaceae), Tagetes patula L. (Asteraceae), and Lobularia maritima (L.) Desv. (Brassicaceae)—as lures and toxic sugar baits as landing markers. Mosquitoes landed only on L. maritima. Freshly cut inflorescences of L. maritima elicited a positive flight response in both sexes of mosquitoes. The analysis of the compounds in the static head space of L. maritima was performed by solid phase microextraction (SPME). Of the single volatile compounds tested, acetophenone was attractive and 1‐octanol caused a flight aversive response. These findings are relevant as there are no reported plant‐derived compounds attractive to A. aegypti. As both the male and female mosquitoes sugar feed, traps baited with plant odors are able to lure the whole adult population, making it an interesting option for including in future mosquito surveillance traps.     

A DREaMR system to simplify combining mutations with rescue transgenes in Aedes aegypti

In most experimental animals, it is challenging to combine mutations and rescue transgenes and to use bipartite systems to assess gene expression. To circumvent the difficulties in combining multiple genetic elements, we developed the DREaMR (Drug-on, REporter, Mutant, Rescue) system. Using Drosophila white as the initial model, we demonstrated that introduction of a single insertion by CRISPR/Cas9 created a null mutation, a tagged rescue construct, which could be induced with doxycycline, and which allowed assessment of protein expression. To create a DREaMR in an organism in which combining multiple genetic elements is more problematic than in Drosophila, we tested the mosquito, Aedes aegypti—the insect vector for dengue, yellow fever, Zika, and other viral diseases. We generated a DREaMR allele in the kh gene, which permitted us to induce expression of the rescue construct, and detect expression of Kh. Thus, this system avoids the need to perform genetic crosses to introduce an inducible rescue transgene in a mutant background, or to combine driver and reporter lines to examine expression of the targeted protein. We propose that DREaMR provides a system that can be applied to additional mosquito vectors as well as other organisms in which CRISPR/Cas9 is effective.     

The ultrastructure of the Aedes aegypti heart

Comparative structural analyses of the heart and associated tissues in 4th instar larvae (L4), pupae and adults of Aedes aegypti were undertaken using a combination of microscopy techniques. The Ae. aegypti heart consists of cardiomyocytes arranged in a helical fashion, and it is physically associated with intersegmental groups of pericardial cells (PCs) and the alary muscles (AMs). Ramifications commonly present in AMs are more developed in adults than in the immature stages. Pericardial cells absorb and store extracellular components as shown by the uptake of carmine dye fed in larval diet. We also observed that carmine stained inclusions corresponding to electron-dense structures resembling lysosomes that were more abundant and prominent in pupae, suggestive of increase of waste accumulation during pupation. The results presented here expand on previously known aspects of the mosquito heart and describe for the first time comparative aspects of the morphology of the heart in different developmental stages.     

Reappraisal of the pore channel system in the grooved pegs of Aedes aegypti

Pore channels occur along the grooves of lactic acid-receptive grooved pegs on the antennae of female Aedes aegypti. There are about 38 pore openings per groove or about 456 per peg. This finding is in conflict with the previous report that pore channels were extremely rare. The pore channels are of a similar electron density to the cuticle of the peg, making them difficult to see. For this reason many of the pore channels were probably overlooked in the previous study. We could not find a terminal pore in the grooved peg as has been reported. Scanning electron microscopy and negative staining of the pegs revealed a tip of variable shape, usually without a pore. It is possible that 'edge effect' (more secondary electrons escape from edges of objects, making them appear brighter than central regions) leads to an apparent terminal pore. Occasionally pegs have a number of small (20-40 nm) pores in the tip region and these might also have been misinterpreted as a terminal pore. Pore channels appear to be the primary means of entry for air-borne stimuli in these grooved pegs.     

Global genetic diversity of Aedes aegypti

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co‐occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub‐Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans‐Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.     

Monoclonal antibodies as probes for Aedes aegypti trypsin

Two murine monoclonal cell lines secreting antibodies against mosquito trypsin were produced by the hybridoma technique. Selected clones of each line were expanded, injected into mice or rats and the immunoglobulins were purified from ascites fluid. Clones of one cell-line (Mab 1–7) recognized only the major 30 kD trypsin from imaginal midguts on SDS-western blots, while trypsin from larval midguts. bovine trypsin and trypsin from other mosquito species were not recognized. This antibody did not distinguish trypsin from 19 strains within the same species, collected around the world. The second line (Mab 11–12) exhibited crossreactivities with other species when applied to a native blot. Analysis of the digestive cycle with these antibodies revealed an increase in immunoreactivity after blood-feeding with both immunoblots and radioimmunoassays, confirming earlier reports based on enzymatic assays.     

Comparative study of three herbal formulations against dengue vectors Aedes aegypti

The efficacy of three formulations (i.e., natural lavender crude, essential oil, and gel) extracted from Lavender angustifolia was tested against vectors of the epidemic dengue virus, Aedesaegypti, to evaluate their larvicidal activity effect. The ethanolic extract of the lavender crude was prepared using a rotary evaporator, while the other extracts, such as essential oil and gel, were obtained from iHerb, a supplier of medicinal herbs in the US. The mortality rate of larvae was evaluated 24 h after exposure. Larvicidal activity of the lavender crude was 91% mortality at 150 ppm, 94% for essential oil at a concentration of 3000 ppm, and 97% for lavender gel at a 1000 ppm. Natural lavender crude was one of the most promising extracts tested against Ae.aegypti larvae, with lethal concentrations at LC₅₀ and LC₉₀ of 76.4 and 174.5 ppm post-treatment. The essential oil had the least effect on mosquito larvae, with LC₅₀ and LC₉₀ reaching 1814.8 and 3381.9 ppm, respectively. The lavender gel was moderately effective against Ae. aegypti larvae, with LC₅₀ and LC₉₀ values reaching 416.3 and 987.7 ppm after exposure. The occurrence of morphological abnormalities in the larvae treated with the three compounds, in turn, resulted in an incomplete life cycle. Therefore, our results indicated that natural lavender crude displayed the highest larvicidal activity against larvae, followed by gel and essential oil. Thus, this study concluded that lavender crude is an effective, eco-friendly compound that can be used as an alternative to chemical products to control vector-borne epidemic diseases.