Productivity of natural and artificial containers for Aedes polynesiensis and Aedes aegypti in four American Samoan villages

Six mosquito species were identified in a survey of containers associated with 347 households in four villages in American Samoa. Aedes polynesiensis Marks (Diptera: Culicidae) and Aedes aegypti (L) were the most abundant species, representing 57% and 29% of the mosquitoes identified. Culex quinquefasciatus (Say), Culex annulirostris (Skuse), Aedes oceanicus (Belkin) and Toxorhynchites amboinensis (Doleschall) were also found. Aedes aegypti and Ae. polynesiensis showed distinct differences in their use of containers, preferring large and small containers, respectively. By contrast with previous studies, Ae. polynesiensis utilized domestic and natural containers with equal frequency, whereas Ae. aegypti continued to be found predominantly in domestic containers. Only 15% of containers holding immature mosquitoes included pupae and fewer than 10 Aedes spp. pupae were found in most containers with pupae. An estimated 2289 Ae. polynesiensis and 1640 Ae. aegypti pupae were found in 2258 containers. The presence of both species in the same container did not affect the mean density of either species for larvae or pupae. Glass jars, leaf axils, tree holes and seashells produced few Aedes spp. pupae in any of the study villages. Overall, 75% of Ae. polynesiensis pupae were found in buckets, ice-cream containers and tyres, with <7% being produced in natural containers, whereas 82% of Ae. aegypti pupae were found in 44-gallon (US) drums ( approximately 166L), buckets and tyres. Source reduction efforts targeting these container types may yield significant reductions in both Ae. polynesiensis and Ae. aegypti populations in American Samoa.     

The effect of Brugia pahangi infection on survival of susceptible and refractory species of the Aedes scutellaris complex

Life table statistics were used to examine the survival functions of filarial susceptible and refractory species of the Aedes scutellaris (Walker) group of mosquitoes, following infection with high and moderate doses of Brugia pahangi (Buckley & Edeson). Survivorship curves and hazard function curves were generated, and the median survival times and the proportions of mosquitoes surviving beyond the extrinsic incubation period of the parasite were determined. In the susceptible populations of Aedes polynesiensis Marks, Ae. pseudoscutellaris (Theobald) and Ae.tabu Ramalingam & Belkin a dose-response relationship was detected between parasite load and mortality. This relationship was characterized by a significant reduction in the proportions of infected female mosquitoes surviving at days 1 and 9 postinfection, reduction in the median survival times and an increase in the hazard rates as the infectious dose increased. The survival of the refractory species, Ae.alcasidi Huang and Ae.katherinensis Woodhill was not significantly affected by the infection. A positive correlation between microfilaraemia in the vertebrate host and parasite load in the susceptible mosquito populations was also observed. Regression analysis of the number of parasites recovered from susceptible mosquitoes at the time of death showed that mosquitoes at highest risk of dying harboured from 11.6 to 19.4 infective larvae when fed on a gerbil with sixty-five microfilariae per 20 microliters blood; this resulted in 34.4-40.2% mortality by day 9 postinfection. A mean number of 32.6-46.9 infective larvae was observed when these populations were exposed to a gerbil with a microfilaraemia of 150 mf/20 microliters and resulted in 72.8% to 80% mortality in these populations. Viable infective larvae were recovered from infected mosquitoes up to 50 days postinfection.     

The relative attractiveness of carbon dioxide and octenol in CDC- and EVS-type light traps for sampling the mosquitoes Aedes aegypti (L.), Aedes polynesiensis Marks, and Culex quinquefasciatus say in Moorea, French Polynesia.

Two dominant day-biting pests and vector species on the island of Moorea in French Polynesia are Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) polynesiensis Marks, major vectors of dengue viruses and Wuchereria bancrofti, respectively. Their surveillance is hindered by a relative lack of attraction to light traps, necessitating the undesirable use of human bait collections with the inherent risks of pathogen transmission. The effectiveness of CDC- and EVS-type light traps baited with olfactory attractants was evaluated for these two Aedes species and the nocturnal Culex (Culex) quinquefasciatus Say in three sites in urban and semi-rural environments on Moorea in October/November 2003. Firstly, four CDC-type traps with light only, light with octenol, light with carbon dioxide (dry ice), and light with octenol plus carbon dioxide were operated continuously over four days with daily rotation to compensate for position effects. Secondly, two CDC- and two EVS-type traps with carbon dioxide or carbon dioxide plus octenol were operated continuously over four days with similar rotation. Variation was found in the numbers of the three species collected at the different sites, reflecting the relative availability of their preferred larval habitats. With the CDC traps in the first trial, the addition of octenol to the light did not significantly increase the collection of any species, the addition of carbon dioxide did significantly increase collection of all three species, while the addition of octenol to the light plus carbon dioxide did not significantly increase the collections further. In the second trial, there was no significant difference in the mean number of Ae. aegypti or Ae. polynesiensis collected in either EVS or CDC traps when baited with carbon dioxide or with octenol added. For Cx. quinquefasciatus, the supplementation with octenol made no significant difference with EVS traps but resulted in significantly reduced collections in CDC traps. Overall, neither trap, however baited, provided large samples when compared with landing/ biting collections at human bait. Only two other species were collected, Culex (Culex) roseni Belkin and Aedes (Aedimorphus) nocturnus (Theobald), the latter being a first record for the island of Moorea and for French Polynesia.     

Filarial susceptibility and effects of Wolbachia in Aedes pseudoscutellaris mosquitoes

The mosquito Aedes pseudoscutellaris (Theobald), a member of the Aedes (Stegomyia) scutellaris complex (Diptera: Culicidae), is an important vector of subperiodic Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae), causing human lymphatic filariasis, on South Pacific islands. Maternal inheritance of filarial susceptibility in the complex has previously been asserted, and larval tetracycline treatment reduced susceptibility; the maternally inherited Wolbachia in these mosquitoes were suggested to be responsible. To investigate the relationship of these two factors, we eliminated Wolbachia from a strain of Ae. pseudoscutellaris by tetracycline treatment, and tested filarial susceptibility of the adult female mosquitoes using Brugia pahangi (Edeson & Buckley). Filarial susceptibility was not significantly different in Wolbachia-free and infected lines of Ae. pseudoscutellaris, suggesting that the Wolbachia in these mosquitoes do not influence vector competence. Crosses between Wolbachia-infected males and uninfected females of Ae. pseudoscutellaris showed cytoplasmic incompatibility (CI), i.e. no eggs hatched, unaffected by larval crowding or restricted nutrient availability, whereas these factors are known to affect CI in Drosophila simulans. Reciprocal crosses between Ae. pseudoscutellaris and Ae. katherinensis Woodhill produced no progeny, even when both parents were Wolbachia-free, suggesting that nuclear factors are responsible for this interspecific sterility.     

Generation of a novel Wolbachia infection in Aedes albopictus (Asian tiger mosquito) via embryonic microinjection

Genetic strategies that reduce or block pathogen transmission by mosquitoes are being investigated as a means to augment current control measures. Strategies of vector suppression and replacement are based upon intracellular Wolbachia bacteria, which occur naturally in many insect populations. Maternally inherited Wolbachia have evolved diverse mechanisms to manipulate host insect reproduction and promote infection invasion. One mechanism is cytoplasmic incompatibility (CI) through which Wolbachia promotes infection spread by effectively sterilizing uninfected females. In a prior field test, releases of Wolbachia-infected males were used to suppress a field population of Culex pipiens. An additional strategy would employ Wolbachia as a vehicle to drive desired transgenes into vector populations (population replacement). Wolbachia-based population suppression and population replacement strategies require an ability to generate artificial Wolbachia associations in mosquitoes. Here, we demonstrate a technique for transferring Wolbachia (transfection) in a medically important mosquito species: Aedes albopictus (Asian tiger mosquito). Microinjection was used to transfer embryo cytoplasm from a double-infected Ae. albopictus line into an aposymbiotic line. The resulting mosquito line is single-infected with the wAlbB Wolbachia type. The artificially generated infection type is not known to occur naturally and displays a new CI crossing type and the first known example of bidirectional CI in Aedes mosquitoes. We discuss the results in relation to applied mosquito control strategies and the evolution of Wolbachia infections in Ae. albopictus.     

Genetics of speciation in the Aedes (Stegomyia) scutellaris subgroup (Diptera:Culicidae). 4. Chromosomal relationships of Aedes cooki with four sibling species

Morphology and behavior of chromosomes and development of testes and sperm were examined in hybrids from interspecific crosses involving Aedes cooki and four sibling species of the Aedes (Stegomyia) scutellaris subgroup of mosquitoes. The degree of abnormality in hybrid spermatogenesis in interspecific crosses involving Aedes cooki males and females of four sibling species paralleled the geographic distributions of these species and the genetic divergence indicated by other genetic studies. Hybrids from crosses involving Aedes malayensis females and Aedes cooki males were characterized by atrophied testes and extensive chromosome breakage. Hybrids from crosses involving Aedes alcasidi females and Aedes cooki males suggested a possible pericentric inversion distinguishing the largest autosome of Aedes alcasidi from that of Aedes cooki. Hybrids from interspecific crosses involving females of Aedes polynesiensis and Aedes pseudoscutellaris and males of Aedes cooki showed high percentages of univalents of the smallest chromosome pair. Hybrid spermatogenesis in two interspecific crosses involving Aedes cooki females differed from results of reciprocal crosses. Data were scant, however, and interpretation was difficult in view of negligible hatch in all interspecific crosses involving Aedes cooki females.     

Bacteriophage WO-B and Wolbachia in natural mosquito hosts: infection incidence, transmission mode and relative density

Bacteriophages of Wolbachia bacteria have been proposed as a potential transformation tool for genetically modifying mosquito vectors. In this study, we report the presence of the WO-B class of Wolbachia-associated phages among natural populations of several mosquito hosts. Eighty-eight percent (22/25) of Wolbachia-infected mosquito species surveyed were found to contain WO-B phages. WO-B phage orf7 sequence analysis suggested that a single strain of WO-B phage was found in most singly (23/24) or doubly (1/1) Wolbachia-infected mosquitoes. However, the single Wolbachia strain infecting Aedes perplexus was found to harbour at least two different WO-B phages. Phylogenetic analysis suggested that horizontal transmission of WO-B phages has occurred on an evolutionary scale between the Wolbachia residing in mosquitoes. On an ecological scale, a low trend of co-transmission occurred among specific WO-B phages within Wolbachia of each mosquito species. Assessment of the density of WO-B phage by real-time quantitative polymerase chain reaction (RTQ-PCR) revealed an average relative density of 7.76 x 10(5)+/- 1.61 x 10(5) orf7 copies per individual mosquito for a single Wolbachia strain infecting mosquitoes, but a threefold higher density in the doubly Wolbachia-infected Aedes albopictus. However, the average combined density of WO-B phage(s) did not correlate with that of their Wolbachia hosts, which varied in different mosquito species. We also confirmed the presence of WO-B-like virus particles in the laboratory colony of Ae. albopictus (KLPP) morphologically, by transmission electron microscopy (TEM). The viral-like particles were detected after purification and filtration of Ae. albopictus ovary extract, suggesting that at least one WO-B-like phage is active (temperate) within the Wolbachia of this mosquito vector. Nevertheless, the idea of utilizing these bacteriophages as transformation vectors still needs more investigation and is likely to be unfeasible.     

Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells

Wolbachia is a maternal transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce viral interference and spread into mosquito vector population makes it possible to develop Wolbachia as a biological control agent for dengue control. While Wolbachia induces resistance to dengue virus in the transinfected Aedes aegypti mosquitoes, a similar effect was not observed in Aedes albopictus, which naturally carries Wolbachia infection but still serves as a dengue vector. In order to understand the mechanism of this lack of Wolbachia-mediated viral interference, we used both Ae. albopictus cell line (Aa23) and mosquitoes to characterize the impact of Wolbachia on dengue infection. A serial of sub-lethal doses of antibiotic treatment was used to partially remove Wolbachia in Aa23 cells and generate cell cultures with Wolbachia at different densities. We show that there is a strong negative linear correlation between the genome copy of Wolbachia and dengue virus with a dengue infection completely removed when Wolbacha density reaches a certain level. We then compared Wolbachia density between transinfected Ae. aegypti and naturally infected Ae. albopictus. The results show that Wolbachia density in midgut, fatbody and salivary gland of Ae. albopictus is 80-, 18-, and 24-fold less than that of Ae. aegypti, respectively. We provide evidence that Wolbachia density in somatic tissues of Ae. albopictus is too low to induce resistance to dengue virus. Our results will aid in understanding the mechanism of Wolbachia-mediated pathogen interference and developing novel methods to block disease transmission by mosquitoes carrying native Wolbachia infections.     

Aedes polynesiensis in the Society Islands: environmental correlates of isoenzyme differentiation

Abstract Isoenzyme genetic differentiation of Aedes polynesiensis mosquitoes in Raiatea island, French Polynesia, was evaluated by two models of population structure based on seven gene-enzyme systems: Ak, Est, Got, Gpi, Hk, Mdh and Pgm. The ecological model tested whether genetic differentiation is congruent with habitat differences. The isolation model evaluated whether genetic differentiation is proportional to geographical distribution. The ecological model found no significant differentiation between populations of Ae.polynesiensis from beach and forest ecotopes, whereas the isolation model was consistent with the data. However, incipient speciation is opposed by the source-sink system of population dynamics in such small neighbouring islands, where Ae.polynensiensis extinction is readily followed by reinvasion causing considerable gene flow between island populations.     

Relative Wolbachia density of field-collected Aedes albopictus mosquitoes in Thailand

Female Aedes albopictus mosquitoes from natural populations of different geographical regions of Thailand were collected and allowed to oviposit to determine relative Wolbachia A and Wolbachia B densities of their offspring (F1) by using real-time quantitative PCR (RTQ-PCR). An important aspect of this work is that all Aedes albopictus mosquitoes were collected from the field. Twenty-seven offspring were from diverse areas of Thailand (Songkhla, Konkaen, Chantaburi, and Kanchanaburi). The range of relative Wolbachia A density in F, mosquitoes was from 0.007 to 1250.78 (bacteria-to-host ratio), whereas relative Wolbachia B densities ranged from 0 to 348.2 (bacteria-to-host ratio). These data are in contrast to those from a previous study that showed a very low amount (less than 0.10) of both relative Wolbachia density types for laboratory strains. The percent transmission of Wolbachia density from mother to each individual offspring cannot be predicted and was not related to the sex of the F1. Obtaining confirmation for variations and unpredictable Wolbachia transmission load raises some concerns about using Wolbachia as a gene-driving system in nature for population replacement if Wolbachia density is involved in cytoplasmic incompatibility in this mosquito.     

我国蚊虫体内感染的Wolbachia的wsp基因序列测定与分析

测定了我国尖音库蚊复合组和白纹伊蚊蚊虫体内感染的Wolbachia株的wsp基因序列。核苷酸和氨基酸的同源性及系统关系分析表明,我国尖音库蚊复合组和白纹伊蚊中Wolbachia株的wsp基因序列与Pip组其它株的核苷酸及氨基酸同源性分别为98%～100%和97%～100%, 属B大组Wolbachia中的Pip组。     

Chromosomal localization and genomic organization of cloned repetitive DNA fragments in mosquitoes (Diptera: Culicidae)

The chromosomal localization and genomic organization of three cloned repetitive DNA fragments (viz., H-76, H-61, and H-19) isolated from theAedes albopictus genome have been examined inAe. albopictus and six otherAedes species:Ae. aegypti, Ae. seatoi, Ae. flavopictus, Ae. polynesiensis, Ae. alcasidi andAe. katherinensis. The results fromin situ and Southern hybridization analyses show that the sequences homologous to cloned repetitive DNA fragments are dispersed throughout the genome in each species. The sequences homologous to these cloned repetitive DNA fragments are also found inHaemagogus equinus, Tripteroides bambusa andAnopheles quadrimaculatus and are dispersed in their genomes. Data indicate divergence in the amount and the structural organization of sequences homologous to these cloned fragments among mosquito species.     

Midgut and salivary gland barriers to La Crosse virus dissemination in mosquitoes of the Aedes triseriatus group

Vector competence for La Crosse virus (LACV) was compared for four species in the Aedes (Protomacleaya) triseriatus group: Ae. triseriatus (Say), Ae. hendersoni Cockerell, Ae. zoosophus Dyar and Knab and Ae. brelandi Zavortink (Diptera: Culicidae). Rates of replication and dissemination of virus in the mosquito hosts were compared and rates of oral transmission of virus to suckling mice were determined. Barriers to virus dissemination which limited the ability of each species to transmit virus were identified. Ae. zoosophus displayed the highest vector competence for LACV. Both infection and transmission rates were high: 99% and 85% respectively; no significant barriers to LACV were found. Disseminated infection of Ae. triseriatus with LACV was controlled primarily be a midgut escape barrier. When virus was introduced directly into the haemocoel, transmission rates were significantly increased (37% v. 79%). Ae. hendersoni showed high susceptibility to LACV infection but a very low rate of oral transmission (7%). Ae. brelandi was also highly susceptible to infection by LACV and transmitted virus at an intermediate rate (27%). Modulation of vector competence in both Ae. hendersoni and Ae. brelandi resulted from a salivary gland escape barrier. As these four species of mosquitoes comprise a closely related monophyletic series, their differences of vector competence for LACV provide an excellent model for studying the genetic basis of the barriers involved.     

Seasonal distribution and species composition of daytime biting mosquitoes

Adults and immatures of Aedes mosquito populations were collected at temperatures between 40 and 44°C (summer), while larvae were collected at 0°C (winter). Major mosquito activities were observed from February to mid-December at various collection sites that yielded high populations of Aedes spp. from May to September, and high populations of Culex spp. and Anopheles spp. from March to September. In June to July, mosquito activity was suspended because the relative humidity was high (70%); a result of the monsoon rains. In August, with temperature ranging from 38 to 42°C, the populations of Culex , Anopheles and Aedes began to increase (36.8, 32.1 and 26.3%, respectively). Population estimates (through standard prototype Centers for Disease Control (CDC) and Biogents (BG)-sentinel) and species composition of Aedes in forest habitats indicated a rapid increase in the populations of Ae. albopictus (52.3%), Ae. aegypti (19.1%) and Ae. vittatus (28.5%) following the rainy season in July. Areas positive for Ae. albopictus had identical population levels and distribution ranges of Ae. vittatus , however, there were no Ae. aegypti in Ae. albopictus areas from August to September. The population level, seasonal distribution, habitat and areas of adult activity marked by global positioning system (GPS) coordinates are being used for reference and for species composition data of Anopheles spp. (2), Culex spp. (10) and Aedes spp. (5) in addition to associated temperature, relative humidity and physico-chemical factors of larval habitat. Global meteorological changes have caused an expansion of the active period, leading to the mosquito's possibility of being a vector of disease increasing, resulting in the spread of dengue fever.     

Linkage relationships of eleven enzyme loci in the Aedes scutellaris group

Linkage relationships of 11 enzyme loci were determined in backcrosses between Aedes polynesiensis and Aedes kesseli. Three linkage groups established were Aat2-Lap2-Me-Sex, Cat-Ao-Pgm-Idh2-Est6, and Gpi-Odh-Pgd. Lap2 and Cat have not been previously mapped in Aedes. Locus order and linkage groups were the same as those observed for seven loci mapped in Aedes aegypti. The significance of the observed similarities in chromosome organization and differences in crossover values among closely related Aedes are discussed.     

Mutualistic Wolbachia infection in Aedes albopictus: accelerating cytoplasmic drive

Maternally inherited rickettsial symbionts of the genus Wolbachia occur commonly in arthropods, often behaving as reproductive parasites by manipulating host reproduction to enhance the vertical transmission of infections. One manipulation is cytoplasmic incompatibility (CI), which causes a significant reduction in brood hatch and promotes the spread of the maternally inherited Wolbachia infection into the host population (i.e., cytoplasmic drive). Here, we have examined a Wolbachia superinfection in the mosquito Aedes albopictus and found the infection to be associated with both cytoplasmic incompatibility and increased host fecundity. Relative to uninfected females, infected females live longer, produce more eggs, and have higher hatching rates in compatible crosses. A model describing Wolbachia infection dynamics predicts that increased fecundity will accelerate cytoplasmic drive rates. To test this hypothesis, we used population cages to examine the rate at which Wolbachia invades an uninfected Ae. albopictus population. The observed cytoplasmic drive rates were consistent with model predictions for a CI-inducing Wolbachia infection that increases host fecundity. We discuss the relevance of these results to both the evolution of Wolbachia symbioses and proposed applied strategies for the use of Wolbachia infections to drive desired transgenes through natural populations (i.e., population replacement strategies).     

Widespread Wolbachia infection in terrestrial isopods and other crustaceans

Wolbachia bacteria are obligate intracellular alpha-Proteobacteria of arthropods and nematodes. Although widespread among isopod crustaceans, they have seldom been found in non-isopod crustacean species. Here, we report Wolbachia infection in fourteen new crustacean species. Our results extend the range of Wolbachia infections in terrestrial isopods and amphipods (class Malacostraca). We report the occurrence of two different Wolbachia strains in two host species (a terrestrial isopod and an amphipod). Moreover, the discovery of Wolbachia in the goose barnacle Lepas anatifera (subclass Thecostraca) establishes Wolbachia infection in class Maxillopoda. The new bacterial strains are closely related to B-supergroup Wolbachia strains previously reported from crustacean hosts. Our results suggest that Wolbachia infection may be much more widespread in crustaceans than previously thought. The presence of related Wolbachia strains in highly divergent crustacean hosts suggests that Wolbachia endosymbionts can naturally adapt to a wide range of crustacean hosts. Given the ability of isopod Wolbachia strains to induce feminization of genetic males or cytoplasmic incompatibility, we speculate that manipulation of crustacean-borne Wolbachia bacteria might represent potential tools for controlling crustacean species of commercial interest and crustacean or insect disease vectors.     

Evolution of Wolbachia-induced cytoplasmic incompatibility in Drosophila simulans and D. sechellia

Abstract.— The intracellular bacterium Wolbachia invades arthropod host populations through various mechanisms, the most common of which being cytoplasmic incompatibility (CI). CI involves elevated embryo mortality when infected males mate with uninfected females or females infected with different, incompatible Wolbachia strains. The present study focuses on this phenomenon in two Drosophila species: D. simulans and D. sechellia . Drosophila simulans populations are infected by several Wolbachia strains, including w Ha and w No. Drosophila sechellia is infected by only two Wolbachia : w Sh and w Sn. In both Drosophila species, double infections with Wolbachia are found. As indicated by several molecular markers, w Ha is closely related to w Sh, and w No to w Sn. Furthermore, the double infections in the two host species are associated with closely related mitochondrial haplotypes, namely si I (associated with w Ha and w No in D. simulans ) and se (associated with w Sh and w Sn in D. sechellia ). To test the theoretical prediction that Wolbachia compatibility types can diverge rapidly, we injected w Sh and w Sn into D. simulans , to compare their CI properties to those of their sister strains w Ha and w No, respectively, in the same host genetic background. We found that within each pair of sister strains CI levels were similar and that sister strains were fully compatible. We conclude that the short period for which the Wolbachia sister strains have been evolving separated from each other was not sufficient for their CI properties to diverge significantly.