Structure of an Odorant-Binding Protein from the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive ""Lid""

Background: The yellow fever mosquito, Aedes aegypti, is the primary vector for the viruses that cause yellow fever, mostly in tropical regions of Africa and in parts of South America, and human dengue, which infects 100 million people yearly in the tropics and subtropics. A better understanding of the structural biology of olfactory proteins may pave the way for the development of environmentally-friendly mosquito attractants and repellents, which may ultimately contribute to reduction of mosquito biting and disease transmission. Methodology: Previously, we isolated and cloned a major, female-enriched odorant-binding protein (OBP) from the yellow fever mosquito, AaegOBP1, which was later inadvertently renamed AaegOBP39. We prepared recombinant samples of AaegOBP1 by using an expression system that allows proper formation of disulfide bridges and generates functional OBPs, which are indistinguishable from native OBPs. We crystallized AaegOBP1 and determined its three-dimensional structure at 1.85 angstrom resolution by molecular replacement based on the structure of the malaria mosquito OBP, AgamOBP1, the only mosquito OBP structure known to date. Conclusion: The structure of AaegOBP1 (= AaegOBP39) shares the common fold of insect OBPs with six alpha-helices knitted by three disulfide bonds. A long molecule of polyethylene glycol (PEG) was built into the electron-density maps identified in a long tunnel formed by a crystallographic dimer of AaegOBP1. Circular dichroism analysis indicated that delipidated AaegOBP1 undergoes a pH-dependent conformational change, which may lead to release of odorant at low pH (as in the environment in the vicinity of odorant receptors). A C-terminal loop covers the binding cavity and this ""lid"" may be opened by disruption of an array of acid-labile hydrogen bonds thus explaining reduced or no binding affinity at low pH.

The use of transcriptional profiles to predict adult mosquito age under field conditions

Age is a critical determinant of an adult female mosquito's ability to transmit a range of human pathogens. Despite its central importance, relatively few methods exist with which to accurately determine chronological age of field-caught mosquitoes. This fact is a major constraint on our ability to fully understand the relative importance of vector longevity to disease transmission in different ecological contexts. It also limits our ability to evaluate novel disease control strategies that specifically target mosquito longevity. We report the development of a transcriptional profiling approach to determine age of adult female Aedes aegypti under field conditions. We demonstrate that this approach surpasses current cuticular hydrocarbon methods for both accuracy of predicted age as well as the upper limits at which age can be reliably predicted. The method is based on genes that display age-dependent expression in a range of dipteran insects and, as such, is likely to be broadly applicable to other disease vectors.

Field prevalence of Wolbachia in the mosquito vector Aedes albopictus

The endosymbiotic bacteria in the genus Wolbachia have been proposed as a potential candidate to deliver pathogen-blocking genes into natural populations of medically important insects. The successful application of Wolbachia in insect vector control depends on the ability of the agent to successfully invade and maintain itself at high frequency under field conditions. Here, we evaluated the prevalence of Wolbachia infections in a field population of the Wolbachia-superinfected mosquito Aedes albopictus. A field prevalence of 100% (n = 1,016) was found in a single population in eastern Thailand via polymerase chain reaction (PCR) testing of Wolbachia both from individual parent females and their corresponding F1 offspring. This is the first report of accurate Wolbachia prevalence in a field population of an insect disease vector. The prevalence of superinfection was estimated to be 99.41%. All single-infected individual mosquitoes (n = 6) were found to harbor group A Wolbachia. For this particular population, none was found to be single-infected with group B Wolbachia. Our results also show that PCR testing of field materials alone without checking F1 offspring overestimated the natural prevalence of single infection. Thus, the confirmation of infection status by means of F1 offspring was critical to the accurate estimates of Wolbachia prevalence under field conditions.

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.

Insect densoviruses may be widespread in mosquito cell lines

A diagnostic PCR assay was designed based on conserved regions of previously sequenced densovirus genomic DNA isolated from mosquitoes. Application of this assay to different insect cell lines resulted in a number of cases of consistent positive amplification of the predicted size fragment. Positive PCR results were subsequently confirmed to correlate with densovirus infection by both electron microscopy and indirect fluorescent antibody test. In each case the nucleotide sequence of the amplified PCR fragments showed high identity to previously reported densoviruses isolated from mosquitoes. Phylogenetic analysis based on these sequences showed that two of these isolates were examples of new densoviruses. These viruses could infect and replicate in mosquitoes when administered orally or parenterally and these infections were largely avirulent. In one virus/mosquito combination vertical transmission to progeny was observed. The frequency with which these viruses were detected would suggest that they may be quite common in insect cell lines.

Replacement of the natural Wolbachia symbiont of Drosophila simulans with a mosquito counterpart

Inherited rickettsial symbionts of the genus Wolbachia occur commonly in arthropods and have been implicated in the expression of parthenogenesis, feminization and cytoplasmic incompatibility phenomena in their respective hosts. Here we use purified Wolbachia from the Asian tiger mosquito, Aedes albopictus, to replace the natural infection of Drosophila simulans by means of embryonic microinjection techniques. The transferred Wolbachia infection behaves like a natural Drosophila infection with regard to its inheritance, cytoskeleton interactions and ability to induce incompatibility when crossed with uninfected flies. The transinfected flies are bidirectionally incompatible with all other naturally infected strains of Drosophila simulans, however, and as such represent a unique crossing type. The successful transfer of this symbiont between distantly related hosts suggests that it may be possible to introduce this agent experimentally into arthropod species of medical and agricultural importance in order to manipulate natural populations genetically.

Does habitat modification affect oviposition by the salt marsh mosquito, Ochlerotatus vigilax (Skuse) (Diptera : Culicidae)?

Studies were conducted at sites in south-cast Queensland, Australia, to investigate the effect of habitat modification for mosquito control on the distribution of eggshells of the salt marsh mosquito, Ochlerotatus vigilax (Skuse). Modifications were mainly tunnelling, but an Open Marsh Water Management (OMWM) site and a grid-ditched site were also included. There were two separate experimental designs: one was data collected Before and After (BA) modification and the other was for other sites with a Treatment and Control (TC) experimental design. For the BA data, there were significant reductions in eggshells after modification. Eggshells were generally fewer after modification in areas which were close to unrestricted tidal flushing. A sandy substrate and vegetation changes which resulted in reduced Sporobolus virginicus or mixed Sporobolus and Sarcocornia quinqueflora also contributed to the effect. In the TC experiment, there was no effect of modification at the tunnelled site, eggshells were fewer at the OMWM site, but there were more eggshells at the grid-ditched site. There was some general indication that recent oviposition activity was reduced in sites that had been modified, evidenced by a relatively small proportion of young (dark coloured) eggshells.

THE TEMPO AND MODE OF EVOLUTION OF TRANSPOSABLE ELEMENTS AS REVEALED BY MOLECULAR PHYLOGENIES RECONSTRUCTED FROM MOSQUITO GENOMES

Although many mathematical models exist predicting the dynamics of transposable elements (TEs), there is a lack of available empirical data to validate these models and inherent assumptions. Genomes can provide a snapshot of several TE families in a single organism, and these could have their demographics inferred by coalescent analysis, allowing for the testing of theories on TE amplification dynamics. Using the available genomes of the mosquitoes Aedes aegypti and Anopheles gambiae, we indicate that such an approach is feasible. Our analysis follows four steps: (1) mining the two mosquito genomes currently available in search of TE families; (2) fitting, to selected families found in (1), a phylogeny tree under the general time-reversible (GTR) nucleotide substitution model with an uncorrelated lognormal (UCLN) relaxed clock and a nonparametric demographic model; (3) fitting a nonparametric coalescent model to the tree generated in (2); and (4) fitting parametric models motivated by ecological theories to the curve generated in (3).

Ecological distribution of mosquito larvae of the Anopheles punctulatus group on Niolam (Lihir) Island, Papua New Guinea

We surveyed the larval habitats of member,, of the Anopheles punctulatus group of mosquitoes on Niolam (Lihir) Island. Papua New Guinea. Identification of this group was undertaken by polymerase chain reaction-restriction fragment length polymorphism analysis of the amplified internal transcribed spacer unit 2 of rDNA. because morphologic separation of member species is unreliable. The most widespread malaria vector species and their most common larval habitats identified to aid source-reduction programs for malaria control. The most ubiquitous species was An. punctulatus, followed by An. farauti no. 2. then An. farauti s.s. Anopheles punctulatus has increased relative to An.farauti s.l. since the start of development projects on Lihir Island. The most common larval habitats were shallow temporary pools with clay substrate and with plants or floatage. These habitats. mostly encountered alongside poorly drained roads, may be increased by development projects.

Mosquito isolates of Ross River virus from Cairns, Queensland, Australia

During 1996-1998 60,619 mosquitoes were collected around Cairns, Australia and processed for Alphavirus isolation. Thirty-three isolates of Ross River (RR) virus were made from 9 species, Aedes imprimens, Aedes kochi, Aedes notoscriptus, Aedes vigilax, Culex annulirostris, Culex gelidus, Mansonia septempunctata, Verrallina (formerly Aedes) carmenti, and Verrallina lineatus. Attempts to isolate RR virus from 121 Aedes aegypti were unsuccessful. Twenty six (79%) of the isolates came from within 1 km of a colony of spectacled flying-foxes, Pteropus conspicillatus. The minimum infection rate for these mosquitoes was 1.0 compared with 0.2 per 1,000 for mosquitoes trapped at all other sites. Ross River virus has not previously been isolated from Ae. imprimens, Cx. gelidus, Ma. septempunctata, Ve. carmenti, or Ve. lineatus. This is also the first isolation of an arbovirus from Cx. gelidus in Australia. In conclusion, the vector status of Ve. carmenti, Ae. aegypti and Mn. septempunctata warrants further study. This study also provides evidence that P. conspicillatus may be a reservoir host.

Japanese encephalitis on Badu Island, Australia: the first isolation of Japanese encephalitis virus from Culex gelidus in the Australasian region and the role of mosquito host-feeding patterns in virus transmission cycles

During investigation of an outbreak of Japanese encephalitis (JE) in the Torres Strait, Australia, in 2000, mosquitoes were collected in Badu Island community and at a newly established communal piggery about 3 km from the community. A total of 94285 mosquitoes, comprising 91240 (96.8%) unengorged females, 1630 (1.7%) blood-engorged females and 1415 (1.5%) males, were processed for virus isolation. One isolate of JE virus was obtained from Culex gelidus, with a minimum infection rate of 12.4:1000. This is the first isolate of JE virus from Cx. gelidus in the Australasian region. No isolates were obtained from Cx. annulirostris, the primary implicated Australian JE vector. Analysis of mosquito host-feeding patterns, using gel diffusion, demonstrated that Cx. annulirostris and 5 other species fed predominately on mammals, Analysis of blood-fed mosquitoes collected within the community demonstrated that the proportion of Cx. annulirostris feeding on pigs in 2000 (2.3%) was significantly lower than that for the 1995-97 period (31.3%). The removal of the pigs from Badu Island community has limited the contact between potential amplifying hosts and mosquitoes, thus potentially reducing the risk of transmission of JE virus to the human population.