Genetic differentiation of the dengue vector, Aedes aegypti (Ho Chi Minh City, Vietnam) using microsatellite markers

Dengue haemorrhagic fever emerged in the 1950s and has become a major public health concern in most Asian countries. In Vietnam, little is known about the intraspecific variation of the vector and its consequences on vectorial capacity. Here we report the use of microsatellite markers to differentiate Aedes aegypti populations in Ho Chi Minh City, a typical, overcrowded Asian city. Six microsatellite loci, with 5-14 alleles per locus, were scored in 20 mosquito samples collected in 1998 in Ho Chi Minh City. We found substantial differentiation among Ae. aegypti populations from the outskirts, whereas populations from the centre of the city showed less differentiation. These results are consistent with the hypothesis that populations of Ae. aegypti in central Ho Chi Minh City are panmictic because there are abundant larval breeding sites and an abundance of humans for adults to feed upon. In contrast, populations on the outskirts become differentiated largely through the processes of genetic drift because larval breeding sites are not as abundant. These findings implicate human activities associated with urbanization, as factors shaping the genetic structure of Ae. aegypti populations.     

Bloodmeal microfilariae density and the uptake and establishment of Wuchereria bancrofti infections in Culex quinquefasciatus and Aedes aegypti.

The relationship between ingestion of microfilariae (mf), production of infective larvae (L3) and mf density in human blood has been suggested as an important determinant in the transmission dynamics of lymphatic filariasis. Here we assess the role of these factors in determining the competence of a natural vector Culex quinquefasciatus and a non vector Aedes aegypti to transmit Wuchereria bancrofti. Mosquitoes were infected via a membrane feeding procedure. Both mosquito species ingested more than the expected number of microfilariae (concentrating factor was 1.28 and 1.81 for Cx. quinquefasciatus and Ae. aegypti, respectively) but Cx. quinquefasciatus ingested around twice as many mf as Ae. aegypti because its larger blood meal size. Ae. aegypti showed a faster mf migration capacity compared to Cx. quinquefasciatus but did not allow parasite maturation under our experimental conditions. Similar proportions of melanized parasites were observed in Ae. aegypti (2. 4%) and Cx. quinquefasciatus (2.1%). However, no relationship between rate of infection and melanization was observed. We conclude that in these conditions physiological factors governing parasite development in the thorax may be more important in limiting vectorial competence than the density of mf ingested.     

Structural organization of posterior midgut muscles in mosquitoes, Aedes aegypti and Anopheles gambiae

In order to adapt to diverse feeding behavior, animal intestines have evolved with distinct differences. Such adaptation may include the structure of the longitudinal and circular muscles that maintain the integrity and the tensile strength of the gut tissue in higher metazoans. Here we examined the structural organization of the posterior midgut muscles of two insects, Aedes aegypti and Anopheles gambiae. We found the estimated number of longitudinal muscles in a cross-section to be 168 in Ae. aegypti and 37 in An. gambiae. Within the region, the estimated number of circular muscles is 77 in Ae. aegypti and 57 in An. gambiae. In An. gambiae, longitudinal muscles appear as sets of parallel bundles. Each set overlaps its neighbor to form a continuous tube. We found that this novel mode of muscle fiber sharing makes all circular muscles interconnected. Both types of muscle lie orthogonally to form a grid that holds the epithelium of the posterior midgut. In Ae. aegypti, the muscle fibers between the bundles are shared extensively, making the organization more intricate. This study implies that, because of its simple structure, the insect midgut may provide a powerful tool with which to study the structural evolution and function of animal intestines.     

Effects of Toxorhynchites moctezuma larval predation on Aedes aegypti populations: experimental evaluation

Abstract. Predatory larvae of the mosquito Toxorhynchites moctezuma were used experimentally to control a standing crop of larvae of the dengue vector mosquito Ae.aegypti. Each week, fifty Ae.aegypti first instar larvae were introduced to each of five water-filled drums (220 litres) of the type commonly used for domestic water storage in Caribbean dwellings. At the beginning of the fourth week, a certain number (0, 1, 2, 5 or 10) of first instar Tx.moctezuma larvae were introduced to each drum and the daily yield of Ae.aegypti adults from each drum was monitored thereafter. The experiment was repeated three times. With only one or two Tx.moctezuma larvae, predation on Ae.aegypti larvae stopped the output of Ae.aegypti adults for 1 week. Five or ten Tx. moctezuma prevented any Ae.aegypti emergence for up to 16 weeks. Cannibalism among Tx. moctezuma larvae was seldom observed and appeared not to be a hindrance in using this species against Ae.aegypti. Thus Tx.moctezuma is regarded as a good candidate for the biological control of Ae.aegypti by augmentative releases.     

Larval growth and biosynthesis of reserves in mosquitoes

Developmental instars of four species of mosquitoes have been analyzed for growth and synthesis of biomass with respect to their caloric content of protein, lipids, and carbohydrates for each instar of Aedes aegypti and Culex pipiens of the subfamily Culicinae, and Anopheles albimanus, and An. gambiae of the subfamily Anophelinae. The diameter of the thorax grows during the intermolt, reflecting continuous increase in biomass because it correlates significantly with the larval synthesis of total protein, lipids, and carbohydrates. For Ae. aegypti the fourth instar was sexed to disclose the sex-specific synthetic potential. In Ae. aegypti the protein increased in linear proportion with larval body size, whereas lipid synthesis followed a significant, exponential regression, which was clearly steeper in male larvae and most pronounced in the last instar. When normalized for size, the size-specific protein and lipid contents showed minimal levels of 0.25 and 0.1, respectively, regardless of standard or crowded rearing conditions. The rate of lipid synthesis in Ae. aegypti was determined by incubating fourth instar larvae with (14)C-acetate and estimating the lipids. The rate was highest in the early larvae and decreased towards the end shortly before pupation; in male larvae incorporation was twice the rate of female larvae. Cx. pipiens reached the largest body sizes of all species tested, with protein and lipids increasing linearly with size. Their minimal levels of size-specific caloric contents were around 0.35 for protein and 0.25 for lipids. Anopheles also showed a linear relationship between larval size and caloric protein and lipid contents. Their minimal threshold levels in size-specific contents were 0.35 for protein and 0.2 for total lipids, similar to Culex, but slightly higher than in Aedes. Starvation of Ae. aegypti larvae and subsequent feeding partially improved their lipid contents, but never to the levels of non-starving, optimal controls. Conversely, well-fed final instars exposed to complete starvation showed a tremendous reduction of the protein and lipids contents in the surviving imagines, accompanied by 73% mortality. These results demonstrate the biosynthetic plasticity and the significance of the phagoperiod in Ae. aegypti during the final fourth instar for growth. The characteristic differences between these two subfamilies in their larval physiology are discussed in relation to ecological factors as encountered in the field under natural conditions, and in relation to our earlier findings on the reproductive physiology.     

Mosquito traps designed to capture Aedes aegypti (Diptera: Culicidae) females: preliminary comparison of Adultrap, MosquiTRAP and backpack aspirator efficiency in a dengue-endemic area of Brazil

In this report, the efficiency of Adultrap under field conditions is compared to a CDC backpack aspirator and to MosquiTRAP. An urban dengue-endemic area of Rio de Janeiro was selected to evaluate the efficiency of mosquito traps in capturing Aedes aegypti females. Adultrap and aspirator captured similar numbers of Ae. aegypti females, with the former showing high specificity to gravid individuals (93.6%). A subsequent mark-release-recapture experiment was conducted to evaluate Adultrap and MosquiTRAP efficiency concomitantly. With a 6.34% recapture rate, MosquiTRAP captured a higher mean number of female Ae. aegypti per trap than Adultrap (chi2 = 14.26; df = 1; p < 0,05). However, some MosquiTRAPs (28.12%) contained immature Ae. aegypti after 18 days of exposure in the field and could be pointed as an oviposition site for female mosquitoes. Both trapping methods, designed to collect gravid Ae. aegypti females, seem to be efficient, reliable and may aid routine Ae. aegypti surveillance.     

Microsatellite isolation and linkage group identification in the yellow fever mosquito Aedes aegypti

Microsatellites have proved to be very useful as genetic markers, as they seem to be ubiquitous and randomly distributed throughout most eukaryote genomes. However, our laboratories and others have determined that this paradigm does not necessarily apply to the yellow fever mosquito Aedes aegypti. We report the isolation and identification of microsatellite sequences from multiple genomic libraries for A. aegypti. We identified 6 single-copy simple microsatellites from 3 plasmid libraries enriched for (GA)(n), (AAT)(n), and (TAGA)(n) motifs from A. aegypti. In addition, we identified 5 single-copy microsatellites from an A. aegypti cosmid library. Genetic map positions were determined for 8 microsatellite loci. These markers greatly increase the number of microsatellite markers available for A. aegypti and provide additional tools for studying genetic variability of mosquito populations. Additionally, most A. aegypti microsatellites are closely associated with repetitive elements that likely accounts for the limited success in developing an extensive panel of microsatellite marker loci.     

Surveillance of the mosquito Aedes aegypti and its biocontrol with the copepod Mesocyclops aspericornis in Australian wells and gold mines

Abstract. A survey of the dengue vector mosquito Aedes aegypti was undertaken using runnel traps to detect immature stages (larvae and pupae) in flooded disused mine shafts and wells in Charters Towers, Queensland, Northern Australia. The town has a history of dengue fever since 1885 when goldminers were the first recorded victims. During the latest dengue epidemic in 1993, 2% of the population had laboratory-confirmed dengue virus Type 2, despite source reduction of Ae.aegypti breeding-sites at ground level or above. This led to suspicions that dengue vector Ae.aegypti breeding-sites might be below ground level. When surveyed in March 1994, Ae.aegypti immatures were found in 9/10 wells and 1/6 mine shafts. The water in wells and mines had similar characteristics -except that turbidity was higher in the mines, which more often contained predators of mosquito immatures.
The copepod Mesocyclops aspericornis was collected from water in 1/10 wells and 2/6 mine shafts. Laboratory predation trials resulted in 95.5–100% predation by 25 copepods/1 on Ae.aegypti first-instar larvae up to 200 larvae/1. Five wells containing Ae.aegypti in the survey were inoculated with fifty indigenous M.aspericornis , and five wells (one positive and four negative in the survey) were left untreated as controls. Nine months later, in December 1994, Ae.aegypti had been eliminated from all five treated wells but all untreated control wells contained Ae.aegypti , except for one well that contained a natural population of M.aspericornis. The role of wells and mines as winter/ dry season refuges of Ae.aegypti in northern Australia is reviewed, and we recommend the use of M.aspericornis as a cost-effective, environmentally acceptable and persistent agent for the sustainable control of Ae.aegypti , especially in inaccessible breeding sites.     

Mosquito larval consumption of toxic arborescent leaf-litter,and its biocontrol potential

Previously we described the mosquito larvicidal properties of decomposed leaf-litter from deciduous trees, especially the alder Alnus glutinosa (L) Gaertn., due to toxic polyphenols and other secondary compounds. To further examine the biocontrol potential of toxic leaf-litter for mosquito control, feeding rates of third-instar mosquito larvae were assessed for examples of three genera: Anopheles stephensi Liston, Aedes aegypti (L) and Culex pipiens L. (Diptera: Culicidae). When immersed in a suspension of non-toxic leaf-litter particles (approximately 0.4 mm), pre-starved larvae of all three species ingested sufficient material in 30 min to fill the anterior gut lumen (thorax plus two to three abdominal segments). Gut filling peaked after 1-2 h ingestion time, filling the intestine up to six to seven abdominal segments for Ae. aegypti, but maxima of five abdominal segments for Cx. pipiens and An. stephensi. Using three methods to quantify consumption of three materials by third-instar larvae of Ae. aegypti, the average amount of leaf-litter (non-toxic 0.4 mm particles) ingested during 3 h was determined as approximately 20 microg/larva (by dry weight and by lignin spectrophotometric assay). Consumption of humine (approximately 100 microm particles extracted from leaf-litter) during 3 h was approximately 80 microg/larva for Ae. aegypti, but only approximately 30 microg/larva for Cx. pipiens and 15 microg/larva for An. stephensi, with good concordance of determinations by dry weight and by radiometric assay. Cellulose consumption by Ae. aegypti was intermediate: approximately 40 microg/larva determined by radiometric assay. Apparent differences between the amounts of these materials ingested by Ae. aegypti larvae (humine four-fold, cellulose two-fold more than leaf-litter) may be attributed to contrasts in palatability (perhaps related to particle size or form), rather than technical discrepancies, because there was good concordance between results of both methods used to determine the amounts of humine and leaf-litter ingested. Bioassays of toxic leaf-litter (decomposed 10 months) with 4-h exposure period (ingestion time) ranked the order of sensitivity: Ae. aegypti (LC50 < 0.03 g/L) > An. stephensi (LC50 = 0.35 g/L) > Cx. pipiens (LC20 > 0.4 g/L). When immersed in the high concentration of 0.5 g/L toxic leaf-litter (0.4 mm particles), as little as 15-30 min ingestion time (exposure period) was sufficient to kill the majority of larvae of all three species, as soon as the gut lumen was filled for only the first few abdominal segments. Possibilities for mosquito larval control with toxic leaf-litter products and the need for standardized ingestion bioassays of larvicidal particles are discussed.     

Comparative efficacy of existing surveillance tools for Aedes aegypti in Western Kenya

All traditional surveillance techniques for Aedes aegypti have been developed for the cosmopolitan domestic subspecies Ae. aegypti aegypti, and not the sylvatic subspecies, Ae. aegypti formosus. The predominant form in Western Kenya is Ae. aegypti formosus that is rarely associated with human habitations but is linked to transmission of sylvatic dengue virus strains. We compared five surveillance methods for their effectiveness in sampling Ae. aegypti formosus with the goal of determining a sustainable surveillance strategy in Kenya. The methods included larval and pupal surveys, oviposition trapping, BG‐Sentinel trapping, resting boxes, and backpack aspirations. Larval and pupal surveys collected the highest number of Ae. aegypti formosus (51.3%), followed by oviposition traps (45.7%), BG‐Sentinel traps (3.0%), and zero collected with either backpack aspiration or resting box collections. No Ae. aegypti formosus larvae or pupae were found indoors. The results indicate that oviposition traps and outdoor larval and pupal surveys were better surveillance methods for Ae. aegypti formosus in Western Kenya.     

Responses of Anopheles gambiae, Anopheles stephensi, Aedes aegypti, and Culex pipiens mosquitoes (Diptera: Culicidae) to cool and humid refugium conditions

Like all terrestrial arthropods, mosquitoes must cope with the threat of desiccation. To gain insight into their survival strategies, we recorded the behavioral responses of Anopheles gambiae, Anopheles stephensi, Aedes aegypti, and Culex pipiens offered zones of different microclimatic conditions in laboratory cages. The cooled refugium was at 25.6 degrees C, 86% RH and the control was at 28.5 degrees C, 75% RH, i.e., a difference in saturation deficit of 3.9 mm Hg between the two zones. We show that newly-emerged adults, with no access to water or sugar, prefer the cooler and more humid refugium with a saturation deficit half that in the control and where the mosquitoes could reduce their metabolic rate. This response is delayed in Ae. aegypti, perhaps because the energy reserves accumulated as larvae are higher in this species. This study shows that mosquitoes under stress can use their thermohygroreceptor cells to guide them to locations that facilitate survival.     

Occurrence of Toxorhynchites guadeloupensis (Dyar Knab) in oviposition trap of Aedes aegypti (L.) (Diptera: Culicidae)

Toxorhynchites guadeloupensis (Dyar Knab), a poorly known mosquito species, was observed preying upon Aedes aegypti (L.) larvae, in an oviposition trap placed for routine dengue entomological surveillance, during 2003-2004 in the urban area of Boa Vista, Roraima, Brazil. This is the first report for Tx. guadeloupensis using Ae. aegypti oviposition traps as breeding places. This finding may have important consequences in the epidemiology and local dengue control since Ae. aegypti density is a basic variable in dengue prediction. Whether predation of Ae aegypti by Tx. guadeloupensis in the Amazon is of significance, is a question to be examined. Also, larval predation may be a cause for underestimation of the actual Ae aegypti numbers. Together these hypotheses need to be better investigated as they are directly related to dengue epidemiology, to the success of any outbreak prediction and surveillance program.     

Parity and ovarian development of Aedes aegypti and Ae. albopictus (Diptera: Culicidae) in metropolitan Rio de Janeiro.

Vector blood-feeding frequency, parity, and ovarian development are important factors that can influence pathogen transmission. Parity rates of the dengue vectors Aedes aegypti and Ae. albopictus were determined from females collected from August 2002 to July 2004 in metropolitan Rio de Janeiro. A high frequency of parous Ae. aegypti (92.9%, n = 550) and Ae. albopictus (99.1%, n = 320) females suggested high survivorship of both species. A total of 69% of wild-caught Ae. aegypti females had blood in the midgut compared to 19% of Ae. albopictus. For Ae. aegypti, red-colored midgut contents were associated with ovaries in early stages of development, and brown-colored midguts were associated with ovaries in late stages of maturation. Ovaries of Ae. aegypti females without blood in the midgut were most frequently in stages I and V of Christophers.     

siRNA-mediated gene targeting in Aedes aegypti embryos reveals that frazzled regulates vector mosquito CNS development

Although mosquito genome projects uncovered orthologues of many known developmental regulatory genes, extremely little is known about the development of vector mosquitoes. Here, we investigate the role of the Netrin receptor frazzled (fra) during embryonic nerve cord development of two vector mosquito species. Fra expression is detected in neurons just prior to and during axonogenesis in the embryonic ventral nerve cord of Aedes aegypti (dengue vector) and Anopheles gambiae (malaria vector). Analysis of fra function was investigated through siRNA-mediated knockdown in Ae. aegypti embryos. Confirmation of fra knockdown, which was maintained throughout embryogenesis, indicated that microinjection of siRNA is an effective method for studying gene function in Ae. aegypti embryos. Loss of fra during Ae. aegypti development results in thin and missing commissural axons. These defects are qualitatively similar to those observed in Dr. melanogaster fra null mutants. However, the Aa. aegypti knockdown phenotype is stronger and bears resemblance to the Drosophila commissureless mutant phenotype. The results of this investigation, the first targeted knockdown of a gene during vector mosquito embryogenesis, suggest that although Fra plays a critical role during development of the Ae. aegypti ventral nerve cord, mechanisms regulating embryonic commissural axon guidance have evolved in distantly related insects.     

Hemocyte monophenol oxidase activity in mosquitoes exposed to microfilariae of Dirofilaria immitis

Monophenol oxidase (MPO) activity in hemocytes collected from Aedes aegypti Liverpool strain and Aedes trivittatus intrathoracically inoculated with saline alone, inoculated with Dirofilaria immitis microfilariae (mff), or from uninoculated mosquitoes was compared using a radiometric tyrosine hydroxylation assay. Hemocyte MPO activity in mff-inoculated (= immune-activated) mosquitoes was significantly increased at 24 hr postinoculation (PI) in A. aegypti and at 6, 12, and 24 hr PI in A. trivittatus as compared with saline-inoculated controls. Baseline and immune-activated levels of hemocyte MPO activity in A. trivittatus were significantly higher compared with those seen in A. aegypti. Baseline hemocyte population levels were similar in both species, but immune activation did not elicit increases in total hemocyte populations in A. trivittatus as has been demonstrated for A. aegypti. Likewise, immune activation by the inoculation of mff did not significantly alter plasma MPO activity in A. trivittatus as compared with uninoculated or saline-inoculated mosquitoes. Plasma MPO activity in A. aegypti, however, appears to constitute a major component of the immune response. The importance of phenol oxidase(s) in the immune response of mosquitoes against mff and the relationship of observed differences in MPO activity to differences in immunological capability between A. aegypti and A. trivittatus are assessed.     

Phylogeography of Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus (Skuse) (Diptera: Culicidae) based on mitochondrial DNA variations

Aedes (Stegomyia) aegypti (l.) and Aedes (Stegomyia) albopictus (Skuse) are the most important vectors of the dengue and yellow-fever viruses. Both took advantage of trade developments to spread throughout the tropics from their native area: A. aegypti originated from Africa and a. albopictus from South-East Asia. We investigated the relationships between A. aegypti and A. albopictus mosquitoes based on three mitochondrial-DNA genes (cytochrome b, cytochrome oxidase I and NADH dehydrogenase subunit 5). Little genetic variation was observed for a. albopictus, probably owing to the recent spreading of the species via human activities. For A. aegypti, most populations from South America were found to be genetically similar to populations from South-East Asia (Thailand and Vietnam), except for one sample from Boa Vista (northern Amazonia), which was more closely related to samples from Africa (Guinea and Ivory Coast). This suggests that African populations of A. aegypti introduced during the slave trade have persisted in Boa Vista, resisting eradication campaigns.     

Characterization of eight single nucleotide polymorphism markers in Aedes aegypti

Sequencing of part of seven genes from Aedes aegypti collected in 16 Brazilian cities revealed the existence of 53 single nucleotide polymorphisms (SNPs), representing one SNP every 52 base pairs. From these 53 SNPs, we selected eight that are independent and highly polymorphic. We describe the use of these markers for differentiation of Brazilian populations of A. aegypti. These are the first SNPs developed for delineating population structure in A. aegypti, and will be a useful complement to epidemiological studies.     

Geographic genetic variation in populations of the dengue virus vector Aedes aegypti

Isoenzyme variation was assessed in 79 mosquito samples of Aedes aegypti, and susceptibility to a dengue 2 virus strain was evaluated in 83 samples. Analysis of FST values, differentiation indexes, and geographic distances separating populations revealed that genetic differences between populations depended on the species' history of migration and colonization. Three major clusters were identified: (1). the sylvan form, Ae. ae. formosus, from West Africa and some islands in the Indian Ocean; (2). the domestic form, Ae. ae. aegypti, from Southeast Asia and South America; and (3). Ae. ae. aegypti populations from the South Pacific islands. Two groups were identified on the basis of susceptibility to the dengue virus: (1). populations with high infection rates, mostly the Ae. ae. aegypti form, and (2). mosquitoes with lower infection rates, specifically Ae. ae. formosus. Other evolutionary and epidemiological implications of the genetic variability of Ae. aegypti are also discussed.     

Observations on the seasonal prevalence and vertical distribution patterns of oviposition by Aedes aegypti (L.) (Diptera: Culicidae) in urban high-rise apartments in Trinidad, West Indies.

The seasonal prevalence and vertical distribution of oviposition of Aedes aegypti were studied for 53 wk in 1999--2000 using modified ovitraps located at several elevations. The ovitraps were positioned both indoors and outdoors in high-rise apartments in the urban township of John John, Port of Spain, Trinidad, West Indies. Of 988 ovitraps, 490 were collected during the months of the wet season, with 404 (84.4%) positive with 18,536 Ae. aegypti eggs. Of 498 ovitraps collected during the dry season, 335 (67.3%) were positive with 12,255 Ae. aegypti eggs. Data from different elevations showed that significantly more eggs were collected at 13-24 m elevations than any other elevation. The results suggest that the invasion of high rise ecosystems by Ae. aegypti can enhance transmission of dengue. This ecological shift in the Ae. aegypti population exploited new habitats associated with human activity, suggesting that strategies should be developed to educate householders as well as creating appropriate vector control measures to prevent future threats of dengue transmission.