Life-shortening Wolbachia infection reduces population growth of Aedes aegypti.

Wolbachia bacteria are being introduced into natural populations of vector mosquitoes, with the goal of reducing the transmission of human diseases such as Zika and dengue fever. The successful establishment of Wolbachia infection is largely dependent on the effects of Wolbachia infection to host fitness, but the effects of Wolbachia infection on the individual life-history traits of immature mosquitoes can vary. Here, the effects of life-shortening Wolbachia (wMelPop) on population growth of infected individuals were evaluated by measuring larval survival, developmental time and adult size of Aedes aegypti in intra- (infected or uninfected only) and inter-group (mixed with infected and uninfected) larval competition assays. At low larval density conditions, the population growth of wMelPop infected and uninfected individuals was similar. At high larval densities, wMelPop infected individuals had a significantly reduced population growth rate relative to uninfected individuals, regardless of competition type. We discuss the results in relation to the invasion of the wMelPop Wolbachia infection into naturally uninfected populations.

Interaction of Wolbachia and Bloodmeal Type in Artificially Infected Aedes albopictus (Diptera: Culicidae).

Maternally inherited Wolbachia bacteria are being introduced into vector mosquito populations, with the goal of reducing the transmission of diseases such as dengue fever. The infection dynamics of Wolbachia depends upon the ability of Wolbachia to manipulate host reproduction as well as any fitness costs imposed upon the host. Some vector mosquito species are opportunistic blood feeders, utilizing both human and nonhuman vertebrate hosts, and the effects of bloodmeal source on Wolbachia phenotype is not well understood. Here we transfer wMelPop Wolbachia from Drosophila melanogaster (Meigen) into wild-type Aedes albopictus (Skuse) and characterize the resulting triple infection by examining for an effect of human and mouse blood on the Wolbachia infection persistence and phenotypes. When provided with human blood, the triple Wolbachia infection was persistent, with high maternal inheritance and relatively little fecundity cost, and a pattern of imperfect unidirectional cytoplasmic incompatibility was observed in mating experiments between wild-type and triply infected individuals. With mouse blood, reduced female fecundity and low maternal inheritance were observed in wMelPop-infected females, which affected the typical pattern of unidirectional CI. Our findings indicate the interactive effects of Wolbachia infection and blood source drive distinct shifts in the Wolbachia-host symbiotic association.

Estimation of population size and dispersal of Aedes polynesiensis on Toamaro motu, French Polynesia.

Mark-release-recapture methods were used to compare Aedes polynesiensis Marks adult numbers and dispersal between dry and wet seasons in a closed population on a small island (motu) in French Polynesia. Females were more than three times more common during wet (December 2008) than dry (May 2007) season samplings although high numbers of vectors were collected during both seasons. Lincoln-Petersen estimates for Ae. polynesiensis females on the motu were 6,055 per hectare for the dry season and 18,860 per hectare for the wet season. Marked females dispersed rapidly to all parts of the motu and survived until recaptures on days 1-5 after release. Males were not adequately sampled using human sentinels or Biogent Sentinel traps.

Monitoring temporal abundance and spatial distribution of Aedes polynesiensis using BG-Sentinel traps in neighboring habitats on Raiatea, Society Archipelago, French Polynesia.

Adult numbers and sizes of mosquitoes were monitored for 2 yr in neighboring habitats on the western coast of Raiatea (Society Archipelago) in anticipation of testing new vector control technologies. Aedes polynesiensis Marks females comprised the overwhelming majority (approximately 99%) of the three species of mosquitoes captured in Biogent Sentinel traps placed at fixed sites on three small satellite islands (motus) of the western lagoon and on the shoreline of Raiatea. Aedes polynesiensis males, Aedes aegypti (L.), and Culex quinquefasciatus Say rarely were collected. Numbers of Ae. polynesiensis females per collection differed among trapping dates and locations, with the majority of females captured on two motus, Horea and Toamaro. Shoreline and Horea females had significantly longer mean wing lengths than females from Tiano and Toamaro. Thus, wing lengths were influenced more by local developmental conditions than overall numbers of adults. Significantly more females were captured during the wet season than the dry season. Nonetheless, at least on the two highly productive motus, dry-season females had larger wing lengths than their wet season counterparts. Local weather patterns predicted about half the variation in mosquito numbers. Differences in vector abundance observed when comparing neighboring motus are likely because of differences in human activity and mosquito suppression.

Costs and benefits of Wolbachia infection in immature Aedes albopictus depend upon sex and competition level.

Bacterial endosymbionts induce various effects on hosts and can dramatically impact host fitness and development. An example is provided by obligate, maternally-inherited Wolbachia, which infect a broad range of invertebrates. Wolbachia are capable of altering host reproduction, thereby promoting infection spread. Wolbachia also pose direct physiological costs and benefits to hosts, complicating their categorization as parasites or mutualists. This study examines for an effect of Wolbachia infection in intra-specific larval competition by Aedes albopictus mosquitoes, with the goal of examining for an impact of Wolbachia infection in mixed populations. Similar to prior work examining for an influence of Wolbachia infection on the fitness of A. albopictus in adults, the results presented here support the hypothesized impact of Wolbachia across all life stages, including immatures. The differential competitiveness of infected larvae detected in our experiments indicates that Wolbachia infected A. albopictus females are less competitive relative to uninfected females when competing under highly competitive conditions. In contrast, under low competitive pressures, infected females experience higher survivorship. Thus, Wolbachia infection shifts from parasitism to mutualism as a function of developmental conditions. Results are discussed in relation to the invasion and persistence of Wolbachia in A. albopictus populations. The results are important to the evolution of stable Wolbachia symbioses, including Wolbachia invasion of an uninfected population. The resulting infection dynamics that occur in an infected population are discussed.

Artificial triple Wolbachia infection in Aedes albopictus yields a new pattern of unidirectional cytoplasmic incompatibility.

Obligately intracellular Wolbachia bacteria infect numerous invertebrates and often manipulate host reproduction to facilitate the spread of infection. An example of reproductive manipulation is Wolbachia-induced cytoplasmic incompatibility (CI), which occurs commonly in insects. This CI has been the focus both of basic scientific studies of naturally occurring invasion events and of applied investigations on the use of Wolbachia as a vehicle to drive desired genotypes into insect populations ("gene drive" or "population replacement" strategies). The latter application requires an ability to generate artificial infections that cause a pattern of unidirectional incompatibility with the targeted host population. A suggested target of population replacement strategies is the mosquito Aedes albopictus (Asian tiger mosquito), an important invasive pest and disease vector. Aedes albopictus individuals are naturally "superinfected" with two Wolbachia types: wAlbA and wAlbB. Thus, generating a strain that is unidirectionally incompatible with field populations requires the introduction of an additional infection into the preexisting superinfection. Although prior reports demonstrate an ability to transfer Wolbachia infections to A. albopictus artificially, including both intra- and interspecific Wolbachia transfers, previous efforts have not generated a strain capable of invading natural populations. Here we describe the generation of a stable triple infection by introducing Wolbachia wRi from Drosophila simulans into a naturally superinfected A. albopictus strain. The triple-infected strain displays a pattern of unidirectional incompatibility with the naturally infected strain. This unidirectional CI, combined with a high fidelity of maternal inheritance and low fecundity effects, suggests that the artificial cytotype could serve as an appropriate vehicle for gene drive.

Vertical oviposition and Lambornella clarki (Ciliophora: Tetrahymenidae) dispersal by Aedes sierrensis (Diptera: Culicidae) in California.

Vertical stratification of treeholes used by mosquitoes may reflect resource quality or result from interspecific competition. Mosquitoes able to monopolize treeholes with optimal resources may be over-represented in the community. Aedes sierrensis, which is well adapted for the Mediterranean climate of California, has evolved in the absence of interspecific competition, so oviposition should reflect resource quality to a large extent. Artificial oviposition traps mounted at four canopy heights facing north or south on trees in a mixed-oak forest at four elevations of the Pacific Coastal Range were used to assess vertical ovipositional preferences by the western treehole mosquito. Natural dispersal of the ciliated protozoan parasite Lambornella clarki was similarly monitored. Gravid Ae. sierrensis showed no vertical stratification during egg laying in traps. Lambornella clarki were naturally dispersed at relatively low frequency into traps and persisted unless eliminated by larval predation. Aedes sierrensis is not currently constrained into occupying a subset of treeholes. However, invasion of its native range by competitive species may alter oviposition patterns.

Pathogenicity of life-shortening Wolbachia in Aedes albopictus after transfer from Drosophila melanogaster.

Maternally inherited Wolbachia bacteria have evolved mechanisms to manipulate the reproduction of their invertebrate hosts, promoting infection spread. A high fitness cost to the host is maladaptive for obligate endosymbionts, and prior studies show rapid selection of new Wolbachia associations toward commensal or mutualistic symbioses. Here, wMelPop Wolbachia is transferred from Drosophila melanogaster into the mosquito Aedes albopictus. Characterization of the resulting strain provides an extreme example of Wolbachia as a pathogen. In addition to reduced longevity and fecundity, abnormally high Wolbachia density is associated with embryonic mortality that masks the typical pattern of cytoplasmic incompatibility. The results are consistent with earlier reports that show unpredictable shifts in the Wolbachia phenotype after interspecific transfer, which can complicate proposed strategies to modify the age structure of medically important vector populations.

Wolbachia infection and resource competition effects on immature Aedes albopictus (Diptera: Culicidae).

Wolbachia pipientis Hertig and Wolbach (Rickettsiales: Rickettsiaceae) are intracellular alpha-proteobacteria that occur naturally in Aedes albopictus (Skuse) (Diptera: Culicidae) and numerous other invertebrates. These endosymbionts can invade host populations by manipulating host reproduction. Wolbachia infections have been shown to impart both costs and benefits to hosts in terms of development, survival, and fecundity. Here, we monitor intraspecific competition among independent cohorts of infected or uninfected larvae. Levels of competition are manipulated by varying initial larval densities and food levels. Although larval density is observed to have major impacts on immature survivorship, sex ratio of eclosing adults, and developmental rates, the Wolbachia infection status had minimal impact on male immatures and no effect on immature females under these experimental conditions. Female and male immatures were observed to respond differently to competitive pressure, with the functional relationships of females and males consistent with scramble and contest competition, respectively. The results are discussed in relation to the evolution of naturally occurring Wolbachia infections in Ae. albopictus (i.e., natural population replacement events) and public health strategies that propose the manipulation of Wolbachia infections in Ae. albopictus populations.

Sexual differences in larval molting rates in a protandrous mosquito (Diptera: Culicidae) species, Aedes sierrensis.

Day-old larval Aedes sierrensis collected from six newly flooded treeholes in northern California were reared individually in the laboratory under simulated field conditions to compare larval developmental rates of males and females. Time to adult eclosion ranged from 133 to 219 d for this generally univoltine, winter-developing species. Males experienced significantly shorter first, second, and third larval instars than females. Females spent significantly less time as fourth instars (whose endpoint is determined by photoperiod). Length of pupal stage was equal for males and females. Time to mean adult eclosion differed among treeholes but was not determined by latitudinal position of treehole. Wing lengths were shorter for males than females in this sexually dimorphic species and also differed significantly among treeholes. Wing lengths were significantly correlated with total developmental time, but females spending more time in the fourth instar did not emerge as larger adults. In natural treeholes, resource utilization during rapid development by Ae. sierrensis males may limit the size and number of females produced from the same cohort if resources are limiting.

Digital image analysis to estimate numbers of Aedes eggs oviposited in containers.

Monitoring reproductive rates in experiments involving aedine mosquitoes is tedious and time demanding. Here, we demonstrate a protocol for rapid estimation of aedine mosquito egg number. The protocol uses ImageJ, a publicly available image analysis program developed at the U.S. National Institutes of Health. The method relies upon the oviposition behavior of Aedes (i.e., ovipositing on a moist substrate instead of water surface) and upon the contrast between dark-colored aedine eggs and a light, uniformly colored paper that is used as an oviposition substrate. The results for 3 Aedes species show that, following the generation of separate calibration curves for each species, the protocol allows for the accurate and repeatable estimation of samples containing hundreds of aedine eggs. We discuss the use of the protocol for monitoring immature aedine populations in both laboratory and field applications.

Spatiotemporal investigation of adult mosquito (Diptera: Culicidae) populations in an eastern Iowa county, USA.

Landscape and climatic factors regulate distributions of mosquitoes (Diptera: Culicidae) over time and space. The anthropogenic control of mosquito populations is often carried out at a local administrative scale, and it is applied based on the relevant agency's experiential knowledge rather than systematic analysis of spatial and temporal data. To address this shortcoming, a spatial and temporal analysis of landscape and climatic parameters in relation to mosquito populations in Black Hawk County, IA, USA, has been carried out. Adult mosquito sampling took place using CDC light traps from May to August 2003 in representative landscapes. Mosquitoes were identified to species level with Aedes trivittatus (Coquillet) and Aedes vexans (Meigen) dominating the collection totals. The best publicly available spatial data on landscape and demographic attributes were collated and included land cover, human census, soils, floodplain, elevation, wetlands, hydrography, roads, and vegetation indices derived from satellite imagery. Spatial processing was carried out to organize landscape attributes for statistical comparison with abundance data from the potentially important West Nile virus (family Flaviviridae, genus Flavivirus, WNV) vector species Ae. vexans and Ae. trivittatus. Landscape parameters shown to be significantly correlated with mosquito counts included soil hydrological properties, presence in floodplain, wetland areas, and deciduous and bottomland forest cover. Data on temperature and precipitation were used to investigate the climatic influence on the temporal occurrence of mosquito population abundances. Late spring rain provided ample moisture for mosquito development, but low temperatures delayed widespread emergence of Ae. trivittatus and Ae. vexans until June 2003. Landscape and climatic impacts on adult mosquito population distributions were demonstrated, and these results could form the basis for the development of a spatiotemporal modeling framework that would inform anthropogenic mosquito control anld vector-borne disease surveillance. A qualitative discussion concerning Culex pipiens (L.) and Culex restuans Theobald is included.

Changes in relative species compositions of biting midges (Diptera: Ceratopogonidae) and an outbreak of Oropouche virus in Iquitos, Peru.

Species compositions of Culicoides paraensis (Goeldi) (Diptera: Ceratopogonidae), the major vector of Oropouche virus to humans in Central and South American urban cycles, and Culicoides insinuatus Ortiz & Leon differed along a northeast-to-southwest transect across Iquitos, Department of Loreto, Peru. The relative distributions of the species were consistent with patterns of human outbreaks along the Amazon River. We resumed collection of biting midges between May 2000 and January 2004 at three sites previously sampled (1996 -1997) to determine whether the known vector was expanding its range relative to the earlier survey. C. paraensis did not replace C. insinuatus across the region surveyed. Instead, C. insinuatus dominated the more southern sites and significantly increased its relative proportion at all three sites. Apparently, microhabitat differences and not range expansion by C. paraensis were responsible for differences in species compositions across the sample sites.

Mosquito (Diptera: Culicidae) development within microhabitats of an Iowa wetland.

Although they provide a number of valuable ecological services, wetlands also may harbor mosquitoes that are vectors of human pathogens. During 2002 and 2003, we measured biological (i.e., abundances of mosquitoes, other insects, and total heterotrophic bacteria, vegetational cover, and dead organic material), chemical (i.e., pH, dissolved nitrate, dissolved nitrite, dissolved phosphate, total alkalinity, and electrical conductivity), and physical (i.e., water temperature, dissolved oxygen, depth, and turbidity) attributes at fixed survey sites in Beaver Valley Wetlands, a small reconstructed palustrine wetland in Black Hawk County, Iowa. The number of immature mosquitoes was significantly correlated with dissolved nitrate and dissolved phosphate concentrations in both years. During the second year of the study, the number of immature mosquitoes was significantly correlated with nonpredators and water turbidity, but not with other measured variables. Independent variables explained 87 and 70% of the variability in mosquito numbers per survey site for the 2 yr of the study, respectively. The most common species of mosquitoes developing in Beaver Valley Wetlands were Aedes vexans (Meigen) Culex territans Walker, Uranotaenia sapphirina (Osten Sacken), and Culex tarsalis Coquillet. We compared relative risks of disease transmission by the mosquitoes developing in wetland microhabitats based upon published species-specific infection rates and propensities to bite humans. The majority of mosquitoes and the greatest potential disease risks were associated with temporary pools, which represented a small proportion of the wetlands. Although relatively few mosquitoes developed in Beaver Valley Wetlands, targeted control efforts could dramatically reduce the numbers of mosquitoes produced with minimal impacts upon nontarget species.

Effects of larval density and predation by Toxorhynchites amboinensis on Aedes polynesiensis (Diptera: Culicidae) developing in coconuts.

Organisms manipulated as biological control agents of disease vectors should tolerate ranges of developmental conditions exploited by their target species. Furthermore, they should reduce numbers of host-seeking vector adults without providing fitness benefits to larval survivors developing among fewer competitors. We studied electrochemistry in rat-chewed coconuts, an important developmental habitat used by Aedes polynesiensis, a vector of lymphatic filariasis. We also studied the effects of larval density and predation by the mosquito Toxorhynchites amboinensis as predators of Ae. polynesiensis. The predators significantly reduced survival rates of Ae. polynesiensis and numbers of males and females developing in coconut husks. Adults from cohorts of Ae. polynesiensis exposed to predators emerged at the same time and were equal in size to adults emerging from predator-free cohorts. No differences were detected in the numbers or sizes of Ae. polynesiensis reaching adulthood among the densities tested. At least for this common natural habitat, Tx. amboinensis gave a good level of biological control of the vector Ae. polynesiensis.

Biting rates and developmental substrates for biting midges (Diptera: Ceratopogonidae) in Iquitos, Peru.

Biting midges (Diptera: Ceratopogonidae) were collected at 16 periurban and rural sites around Iquitos, Peru, between 17 October 1996 and 26 May 1997. Culicoides paraensis (Goeldi), the principal vector of Oropouche virus, was the most commonly collected species (9,086 flies) with Culicoides insinuatus Wirth & Blanton second (7,229 flies). Although both species were collected at all sampling sites (linear (distance surveyed approximately 25 km), C. paraensis dominated at northern collection sites (> 90%), whereas C. insinuatus prevailed at southern collection sites (> 60%). C. paraensis were collected from human sentinels at a constant rate throughout daylight hours, at similar rates during wet and dry months, and regardless of rainfall. Larval developmental substrates for C. paraensis included decaying platano (Musa x paradisiaca L. [Musaceae]) stems, stumps, flowers, fruits, and debris beneath platano trees as well as from soil beneath a fruiting mamay (Syzygium malaccense Merr. & Perry [Myrtaceae] ) tree and organic-rich mud along a lake shoreline. C. insinuatus adults likewise emerged from decaying platano and organic-rich mud along a lake shoreline, but also from debris accumulated in the axils of aguaje (Mauritia flexuosa L. [Palmae]) fronds and decaying citrus fruit. Despite high numbers of biting adults near putative substrates, adults of neither species emerged from other decomposing plant material, soil, phytotelmata, or artificial containers. Because both species of biting midges emerged in high numbers from all parts of platano (ubiquitous in Iquitos), it will be challenging to control them through sanitation.