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Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae.

Aedes aegypti densovirus (AeDNV) is a small DNA virus that has been developed into an expression and transducing vector for mosquitoes [Afanasiev et al. (1994) Exp Parasitol 79: 322-339; Afanasiev et al. (1999) Virology 257: 62-72; Carlson et al. (2000) Insect Transgenesis: Methods and Applications (Handler, A.M. & James, A.A., eds), pp. 139-159. CRC Press, Boca Raton]. Virions carrying a recombinant genome expressing the GFP gene were used to characterize the pathogenesis of the virus in 255 individual Aedes aegypti larvae. The anal papillae of the larvae were the primary site of infection confirming previous observations (Afanasiev etal., 1999; Allen-Muira et al. (1999) Virology 257: 54-61). GFP expression was observed in most cases to spread from the anal papillae to cells of the fat body, and subsequently to many other tissues including muscle fibers and nerves. Infected anal papillae were also observed to shrink, or melanize and subsequently fall off in a virus dependent manner. Three to four day-old larvae were less susceptible to viral infection and, if infected, were more likely to survive into adulthood, with 14% of them still expressing GFP as adults. Higher salt concentrations of 0.10-0.15 M inhibited viral infection. Anopheles gambiae larvae also showed infection of the anal papillae (17%) but subsequent viral dissemination did not occur. The persistence of the reporter gene expression into adulthood of Aedes aegypti indicates that transduction of mosquito larvae with recombinant AeDNV may be a means of introducing a gene of interest into a mosquito population for transient expression.

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Codon preference of Aedes aegypti and Aedes albopictus.

The codon bias of two Aedes mosquito species was examined using a sign test. In general, there appeared to be some preference for C + G at the third base position, although this was not statistically significant. While amino acids such as phenylalanine and tyrosine clearly displayed biases, others such as valine and serine appeared to have little or no bias for any particular codon. Three homologous genes of Aedes aegypti and Drosophila melanogaster were compared using the chi-square test and the codon bias of the two species compared. Drosophila melanogaster was found to have a much stronger bias for C + G at the third base position compared to Aedes. The implications and usefulness of the codon bias are discussed.

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Frequent occurrence of IgE and IgG4 antibodies against saliva of Aedes communis and Aedes aegypti mosquitoes in children.

We examined the prevalence of IgE and IgG4 class antibodies to the saliva of Aedes communis and Aedes aegypti mosquitoes in the sera of three groups of exposed children using a sensitive immunoblot method. The frequencies of IgE antibodies to the major 36-kD A. communis and A. aegypti saliva antigens ranged from 82 to 90% in the 20 Finnish, 17 Kenyan, and 20 Mexican children. The corresponding IgG4 antibody frequencies were 85, 41, and 20%, respectively. The nonexposed 20 Icelandic children did not show IgE or IgG4 antisaliva antibodies. Several of the Finnish children showed also IgE and IgG4 antibodies to a 22-kD A. communis saliva antigen. The Finnish children abnormally sensitive to mosquito bites had frequently IgE and IgG4 antibodies to the 22-kD A. communis saliva antigen, suggesting that these antibodies play a role in the pathogenesis of immediate cutaneous mosquito bite reactions. In contrast to this, no increase was found in the A. aegypti antibody frequencies in the Kenyan and Mexican children with papular urticaria, suggesting that humoral immune response to A. aegypti saliva is not involved in the development of this disorder. The present results show that humoral IgE and IgG4 immune responses to Aedes mosquito saliva antigens is common in children living both in temperature and tropical zones. The IgE antibodies seem to be involved in the immediate mosquito bite whealing, and the occurrence of the IgG4 subclass antisaliva antibodies might be an indicator of intense mosquito bite exposure.

Adolescent↗

A simple parafilm M-based method for blood-feeding Aedes aegypti and Aedes albopictus (Diptera: Culicidae).

A simple device for blood-feeding Aedes (Stegomyia) mosquitoes is described. Parafilm M is stretched and pressed into fiberglass window screen to form a packet for holding warmed blood. The method has been used successfully to maintain Aedes albopictus colonies for >2 yr. In typical blood-feeding trials, 53 +/- 0.8% (+/-SE) of Aedes aegypti (Linnaeus) and 31 +/- 6% of Ae. albopictus (Skuse) fed to repletion.

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Integration of Bacillus thuringiensis H-14 formulations and pyriproxyfen for the control of larvae of Aedes aegypti and Aedes albopictus.

Studies were carried out on the bioefficacy and residual activity of Bacillus thuringiensis israelensis H-14 (Bti) (water-dispersible granules of VectoBac ABG 6511 and liquid formulations of VectoBac 12AS) and pyriproxyfen (insect growth regulator, Sumilarv 0.5%) as direct applications for control of larvae of Aedes aegypti and Aedes albopictus. Two dosages of each Bti formulation (285 and 570 international toxic units [ITU]/liter) and the integration of both Bti formulations and pyriproxyfen were used for residual tests with 45-liter earthen jars for a period of 4 wk. In 1 test series, the treated water was replenished daily with 6 liters of seasoned untreated water. In the 2nd test series, the water in the jars was topped up to the 40-liter level during evaluation. Neither Bti formulation remained effective for a full week. Water-dispersible Bti granules provided effective initial control activity against Ae. aegypti and Ae. albopictus for both test designs (with replenishment and without replenishment of water). The higher dosage (570 ITU/liter) for both Bti formulations was only partially effective at the end of 1 wk after being diluted. After 1 wk, water-dispersible Bti granules provided greater larval mortality than did liquid Bti formulation against both mosquito species when integrated with pyriproxyfen. Pyriproxyfen (79.5 and 159 mg/liter) on its own showed low larvicidal activity but provided very effective control of adult emergence. In this study, integration of Bti (285 and 570 ITU/liter) with pyriproxyfen (79.5 mg/liter) extended the duration of partial larval control somewhat, but live larvae persisted throughout the 4-wk test. The integration effect was more obvious when water-dispersible Bti granules were integrated with pyriproxyfen than when liquid Bti was used. Integration of Bti with pyriproxyfen had a negative effect on adult emergence, which was completely inhibited by pyriproxyfen after day 1. Daily replenishment of water increased Bti activity and provided slightly better larval control. Aedes albopictus and Ae. aegypti were both completely susceptible to the higher concentration of Bti and pyriproxyfen in both test designs (with replenishment and without replenishment of water).

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Bloodmeal sources of Aedes triseriatus and Aedes vexans in a southern Wisconsin forest endemic for La Crosse encephalitis virus.

The micro enzyme-linked immunosorbent assay (ELISA) was used to specifically identify bloodmeal sources of Aedes triseriatus Say and Aedes vexans Meigen collected at a site endemic for La Crosse (LAC) encephalitis virus. Deer were the source of 65% of Ae. triseriatus and 94% of Aedes vexans bloodmeals, respectively. Chipmunks and tree squirrels, which are considered to be the major vertebrate amplifying hosts of LAC virus, were the sources of 8% and 16%, respectively, of the bloodmeals of Ae. triseriatus, the vector of LAC virus. The relatively small proportion of vector bloodmeals taken from the amplifying hosts raises further doubts as to the significance of vertebrate amplification in perpetutation of La Crosse virus in nature, i.e. whether vertebrate amplification alone is sufficient to make up for the shortfall of virus infection that occurs during vertical transmission.

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Monitoring of dengue viruses in field-caught Aedes aegypti and Aedes albopictus mosquitoes by a type-specific polymerase chain reaction and cycle sequencing.

Virologic surveillance for dengue through the detection of the prevalent serotype(s) circulating in the human population during inter- and intra-epidemic periods constitutes a reliable sentinel system for dengue outbreaks. We have applied a rapid and sensitive, semi-nested, reverse transcription-polymerase chain reaction (RT-PCR) assay using nonstructural protein 3 gene primers for the type-specific-detection of dengue viruses in artificially infected and in field-caught adult Aedes mosquitoes. In laboratory experiments, the assay was sensitive enough to detect one virus-infected mosquito head in pools of up to 59 uninfected heads. In a prospective field study conducted from April 1995 to July 1996, female adult Ae. aegypti and Ae. albopictus mosquitoes were caught from selected dengue-sensitive areas in Singapore and assayed by RT-PCR. Approximately 20% of 309 mosquito pools were positive for dengue viruses. Of the 23 RT-PCR-positive Ae. aegypti pools (containing 1-17 mosquitoes each), 18 pools (78.3%) were positive for dengue 1 virus. There were 40 RT-PCR-positive Ae. albopictus pools (containing 1-33 mosquitoes each) of which 31 (77.5%) were positive for dengue 1 virus. The predominant virus type responsible for the current dengue epidemic since 1995 was also dengue 1. The geographic locations of the virus-infected mosquitoes correlated with the residences or workplaces of patients within dengue outbreak areas. A total of 43.5% of the positive Ae. aegypti pools and 25.0% of the positive Ae. albopictus pools contained only a single mosquito. Both Aedes species showed similar overall minimum infection rates of 57.6 and 50 per 1,000 mosquitoes. Infected Ae. aegypti were detected as early as six weeks before the start of the dengue outbreaks in 1995 and 1996. However, infected Ae. albopictus appeared later, when the number of cases was increasing. Virologic surveillance by RT-PCR for detecting dengue virus-infected Aedes mosquitoes in the field may serve as an early warning monitoring system for dengue outbreaks.

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A mosquito densovirus infecting Aedes aegypti and Aedes albopictus from Thailand.

A previously undescribed mosquito densovirus was detected in colonies of Aedes aegypti and Ae. albopictus from Thailand, using a polymerase chain reaction (PCR)-based assay. Phylogenetic analysis of this virus showed it to be most closely related to ADNV isolated from Russian Ae. aegypti. Both Aedes species were susceptible to oral infection with the Thai-strain virus. Larval mortality for Ae. albopictus was higher (82%) than for Ae. aegypti (51%). Aedes aegypti were able to transmit the virus vertically to a high (58%) proportion of G1 progeny, and the virus was maintained persistently for up to six generations. A PCR survey of adult Ae. aegypti and Ae. albopictus in Thailand indicated that only Ae. aegypti are infected in the field, with an overall prevalence of 44%. Densovirus infection in adult Ae. aegypti showed distinct seasonal variation. The Thai strain densovirus may play a role in structuring Ae. albopictus and Ae. aegypti populations in nature.

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Key characters for identifying Aedes bahamensis and Aedes albopictus in North America, north of Mexico.

Aedes bahamensis, a species recently introduced into southern Florida represents the first member of the subgenus Howardina to be found in the United States. Its separation from all other Nearctic Aedes is the subject of this work, integrating it into the North American mosquito keys (Darsie and Ward 1981). The key revisions presented are expanded to include the other exotic species now found in the United States, Aedes albopictus.

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Hybridization between Aedes simpsoni and Aedes woodi with observations on the genetic basis of morphological differences.

The public health importance of the genus Aedes makes it imperative that a better understanding of the genetic differences and isolating barriers between the species be developed. Aedes simpsoni and Aedes woodi from near Dar es Salaam, Tanzania, were used in this investigation. Analysis of F(1), F(2), and backcross progeny obtained from forced matings indicates that single factors form the genetic basis of some of the differences between the two species. There is evidence that Ae. simpsoni and Ae. woodi are separated by both premating and postmating reproductive isolating mechanisms.

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Identification of damaged adult female specimens of Aedes albopictus and Aedes aegypti in the New World.

Two introduced species of Aedes (Stegomyia), Aedes albopictus and Aedes aegypti, occur in the New World. Three characters, easily and simultaneously observed from an anteroventral view, allow for rapid and reliable specific identification of most specimens that lack the characteristic scutal scale patterns. These 3 characters are the presence or absence of pale scales on the clypeus; the presence or absence of a narrow, median line of pale scales on the anterior face of the midfemora; and the pattern of pale and dark scales on abdominal sterna III-V.

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Pathogenicity of the Aedes albopictus parvovirus (AaPV), a denso-like virus, for Aedes aegypti mosquitoes.

AaPV, a denso-like virus isolated from a C6/36 clone of the Aedes albopictus cell line, proved to be very pathogenic for Aedes aegypti first and third instar larvae following per os infection. The mortality reached 90% in 10 days for larvae infected at the first instar. Several factors, such as temperature, larval density and stage, and duration of contact with infectious particles, influenced infection. The virus titer in females surviving infection at the third larval instar reached 10(8) TCID50. Adult mosquitoes were sensitive to virus inoculation and to cytotransfection by viral DNA. Histological and ultrastructural studies revealed the presence of dense nuclei in almost all of the larval tissues with the exception of the midgut.

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Comparison of lipid composition of Aedes aegypti and Aedes albopictus cells obtained from logarithmic and stationary phases of growth.

The total lipids, total neutral lipids, and total phospholipids from Aedes aegypti and aedes albopictus cells cultivated in vitro in a medium containing fetal calf serum were analyzed. The mosquito cells were harvested in the logarithmic and stationary phases of growth. The fatty acid profiles of the lipids showed differences during the aging of the cells but not betweeen species. There was an increase in chain elongation and unsaturation of the fatty acids in the stationary phase when compared with the logarithmic phase of growth. The major components of the fatty acid profiles of the cells were 16:0, 16:1, and 18:1 fatty acids. Few similarities were found between the lipid analysis of the mosquito cells and the growth medium.

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Comparison of phospholipid composition of Aedes aegypti and Aedes albopictus cells obtained from logarithmic and stationary phases of growth.

Aedes aegypti and Aedes albopictus cells were grown in tissue culture and harvested at logarithmic and stationary phases of development. The phospholipids were extracted, separated into lipid classes, and fatty acid composition of each fraction determined. The phosphatidylethanolamine fraction was the major lipid (42-54%). With aging, the A. aegypti cells showed an increase in polyenes in the phosphatidylcholine and phosphatidylethanolamine fractions and in monoenes and polyenes in the phosphatidylinositol fraction. The lysophosphatidylcholine fraction had an increase in chain length of the fatty acids with aging of the A. aegypti cells. The A. albopictus cells, with aging, showed increases in chain length and in the relative percentage of polyenes in the lysophosphatidylcholine, phosphatidylcholine, and phosphatidylserine fractions. In the phosphatidylinositol fraction, chain elongation of fatty acids occurred as the cells aged. In the ceramide phosphorylcholine fraction, there were increases in saturation and chain elongation of the fatty acids from the logarithmic to the stationary phase of the A. albopictus cells. An increase in polyenes was observed with aging of the cells in the phosphatidylethanolamine fraction.

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A parvo-like virus persistently infecting a C6/36 clone of Aedes albopictus mosquito cell line and pathogenic for Aedes aegypti larvae.

We have isolated and partially characterized from an apparently healthy C6/36 subclone of Aedes albopictus cell line a small icosahedral non-enveloped DNA virus, designated AaPV. This virus proved to be highly pathogenic for Aedes aegypti neonate larvae. Viral infection persisted for over 4 years in the cell culture without any cytopathic effect. Attempts to infect suckling mice, Drosophila melanogaster adults and Spodoptera littoralis larvae with AaPV were unsuccessful. Similarly, the AaPV failed to replicate in vertebrate and Drosophila cell lines. Virions, about 22 nm in diameter, had a buoyant density of 1.43 g/cm3 and contained three capsid polypeptides with molecular weights of 53, 41 and 40 kDa. A preliminary study of the viral genome indicated the presence of single-stranded DNA. By its biophysical and biochemical properties, this virus appears to be related to the genus Densovirus within the family Parvoviridae, but lacks serological relationships with the other members of this genus.

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The expression of chloramphenicol acetyltransferase in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes using a double subgenomic recombinant Sindbis virus.

Genomic RNA was transcribed in vitro from the double subgenomic recombinant Sindbis (SIN) virus expression vector, pTE/3'2J/CAT, and transfected into BHK-21 cells to generate recombinant virus stocks. TE/3'2J/CAT virus was used to infect C6/36 (Aedes albopictus) cells and adult female Aedes triseriatus. When C6/36 cells were infected with TE/3'2J/CAT virus at a multiplicity of infection (MOI) of greater than 20, 100% of the cells expressed CAT. The number of CAT polypeptides expressed per cell at 24 h post infection (pi) was 8.3 x 10(5). Approximately 4.0 log10TCID50 of the TE/3'2J/CAT virus was intrathoracically inoculated into adult female mosquitoes. Titers greater than 6.0 log10TCID50/ml were detected within 4 days pi and declined to less than 4.0 log10TCID50/ml 20 days following inoculation. CAT activity was detected within 2 days (8 x 10(-5) units of CAT/mosquito or 1.4 x 10(10) CAT polypeptides), peaked at day 6 (4 x 10(-3) units of CAT/mosquito or 7.2 x 10(11) CAT polypeptides), and remained at peak levels to day 20. Immunofluorescence and CAT activity assays were used to localize CAT expression in infected mosquitoes and demonstrated that CAT was present in neural, midgut, ovarian, and salivary gland tissues. Alphavirus-based expression vectors should be useful for expressing heterologous genes in mosquito cells as well as adult mosquitoes.

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Susceptibility of Aedes aegypti and Aedes albopictus larvae to Ascogregarina culicis and Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) from Florida.

The susceptibility of Aedes aegypti to Ascogregarina culicis and Aedes albopictus to Ascogregarina taiwanensis was examined with mosquito and parasite strains from Tampa, FL. When each host was bioassayed with its natural gregarine, the infection intensity indicated that Ae. aegypti was 59% more susceptible to A. culicis (87 gamonts/larva) than Ae. albopictus to A. taiwanensis (47 gamonts/larva). Infections in single and mixed host populations exposed to 100 oocysts/larva of one and both parasites demonstrated that Ae. aegypti harbors higher A. culicis gamont loads than Ae. albopictus of A. taiwanensis. In dual gregarine exposures of single host populations, the A. culicis infection intensity in Ae. aegypti was reduced by approximately 50%. A. taiwanensis exhibited the same capability of infecting Ae. albopictus in single and dual exposures. In mixed host populations there were no cross infections, but A. taiwanensis in Ae. albopictus produced an infection intensity of approximately 70% lower than that of A. culicis in Ae. aegypti.

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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.

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