River Boats Contribute to the Regional Spread of the Dengue Vector Aedes aegypti in the Peruvian Amazon

Background and Objectives

The dramatic range expansion of the dengue vector Aedes aegypti is associated with various anthropogenic transport activities, but little is known about the underlying mechanisms driving this geographic expansion. We longitudinally characterized infestation of different vehicle types (cars, boats, etc.) to estimate the frequency and intensity of mosquito introductions into novel locations (propagule pressure).

Methods

Exhaustive adult and immature Ae. aegypti collections were performed on six different vehicle types at five ports and two bus/ taxi departure points in the Amazonian city of Iquitos, Peru during 2013. Aquatic vehicles included 32 large and 33 medium-sized barges, 53 water taxis, and 41 speed boats. Terrestrial vehicles sampled included 40 buses and 30 taxis traveling on the only highway in the region. Ae. aegypti adult infestation rates and immature indices were analyzed by vehicle type, location within vehicles, and sampling date.

Results

Large barges (71.9% infested) and medium barges (39.4% infested) accounted for most of the infestations. Notably, buses had an overall infestation rate of 12.5%. On large barges, the greatest number of Ae. aegypti adults were found in October, whereas most immatures were found in February followed by October. The vast majority of larvae (85.9%) and pupae (76.7%) collected in large barges were produced in puddles formed in cargo holds.

Conclusions

Because larges barges provide suitable mosquito habitats (due to dark, damp cargo storage spaces and ample oviposition sites), we conclude that they likely serve as significant contributors to mosquitoes’ propagule pressure across long distances throughout the Peruvian Amazon. This information can help anticipate vector population mixing and future range expansions of dengue and other viruses transmitted by Ae. aegypti.     

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.     

Lightning-caused disturbance in the Peruvian Amazon

We surveyed seven lightning strike sites in the northern Peruvian Amazon. An average of 17.3 trees were damaged per strike; large trees (> 60 cm diameter) were disproportionately affected. The results contribute to a growing body of evidence that lightning is an important agent of disturbance pantropically. Abstract in Spanish is available with online material.     

Insecticide-Driven Patterns of Genetic Variation in the Dengue Vector Aedes aegypti in Martinique Island

Effective vector control is currently challenged worldwide by the evolution of resistance to all classes of chemical insecticides in mosquitoes. In Martinique, populations of the dengue vector Aedes aegypti have been intensively treated with temephos and deltamethrin insecticides over the last fifty years, resulting in heterogeneous levels of resistance across the island. Resistance spreading depends on standing genetic variation, selection intensity and gene flow among populations. To determine gene flow intensity, we first investigated neutral patterns of genetic variability in sixteen populations representative of the many environments found in Martinique and experiencing various levels of insecticide pressure, using 6 microsatellites. Allelic richness was lower in populations resistant to deltamethrin, and consanguinity was higher in populations resistant to temephos, consistent with a negative effect of insecticide pressure on neutral genetic diversity. The global genetic differentiation was low, suggesting high gene flow among populations, but significant structure was found, with a pattern of isolation-by-distance at the global scale. Then, we investigated adaptive patterns of divergence in six out of the 16 populations using 319 single nucleotide polymorphisms (SNPs). Five SNP outliers displaying levels of genetic differentiation out of neutral expectations were detected, including the kdr-V1016I mutation in the voltage-gated sodium channel gene. Association tests revealed a total of seven SNPs associated with deltamethrin resistance. Six other SNPs were associated with temephos resistance, including two non-synonymous substitutions in an alkaline phosphatase and in a sulfotransferase respectively. Altogether, both neutral and adaptive patterns of genetic variation in mosquito populations appear to be largely driven by insecticide pressure in Martinique.     

Co-occurrence Patterns of the Dengue Vector Aedes aegypti and Aedes mediovitattus, a Dengue Competent Mosquito in Puerto Rico

Aedes aegypti is implicated in dengue transmission in tropical and subtropical urban areas around the world. Ae. aegypti populations are controlled through integrative vector management. However, the efficacy of vector control may be undermined by the presence of alternative, competent species. In Puerto Rico, a native mosquito, Ae. mediovittatus, is a competent dengue vector in laboratory settings and spatially overlaps with Ae. aegypti. It has been proposed that Ae. mediovittatus may act as a dengue reservoir during inter-epidemic periods, perpetuating endemic dengue transmission in rural Puerto Rico. Dengue transmission dynamics may therefore be influenced by the spatial overlap of Ae. mediovittatus, Ae. aegypti, dengue viruses, and humans. We take a landscape epidemiology approach to examine the association between landscape composition and configuration and the distribution of each of these Aedes species and their co-occurrence. We used remotely sensed imagery from a newly launched satellite to map landscape features at very high spatial resolution. We found that the distribution of Ae. aegypti is positively predicted by urban density and by the number of tree patches, Ae. mediovittatus is positively predicted by the number of tree patches, but negatively predicted by large contiguous urban areas, and both species are predicted by urban density and the number of tree patches. This analysis provides evidence that landscape composition and configuration is a surrogate for mosquito community composition, and suggests that mapping landscape structure can be used to inform vector control efforts as well as to inform urban planning.     

Geographic and ecological distribution of the dengue and chikungunya virus vectors Aedes aegypti and Aedes albopictus in three major Cameroonian towns

Aedes albopictus (Diptera: Culicidae) was first reported in Central Africa in 2000, together with the indigenous mosquito species Aedes aegypti (Diptera: Culicidae). Because Ae. albopictus can also transmit arboviruses, its introduction is a public health concern. We undertook a comparative study in three Cameroonian towns (Sahelian domain: Garoua; equatorial domain: Douala and Yaoundé) in order to document infestation by the two species and their ecological preferences. High and variable levels of pre‐imaginal Ae. aegypti and Ae. albopictus infestation were detected. Only Ae. aegypti was encountered in Garoua, whereas both species were found in Douala and Yaoundé, albeit with significant differences in their relative prevalence. Peridomestic water containers were the most strongly colonized and productive larval habitats for both species. No major differences in types of larval habitat were found, but Ae. albopictus preferentially bred in containers containing plant debris or surrounded by vegetation, whereas Ae. aegypti tended to breed in containers located in environments with a high density of buildings. These findings may have important implications for vector control strategies.     

Determinants of Heterogeneous Blood Feeding Patterns by Aedes aegypti in Iquitos,Peru

Background

Heterogeneous mosquito biting results in different individuals in a population receiving an uneven number of bites. This is a feature of many vector-borne disease systems that, if understood, could guide preventative control efforts toward individuals who are expected to contribute most to pathogen transmission. We aimed to characterize factors determining biting patterns of Aedes aegypti, the principal mosquito vector of dengue virus.

Methodology/Principal Findings

Engorged female Ae. aegypti and human cheek swabs were collected from 19 houses in Iquitos, Peru. We recorded the body size, age, and sex of 275 consenting residents. Movement in and out of the house over a week (time in house) and mosquito abundance were recorded on eight separate occasions in each household over twelve months. We identified the individuals bitten by 96 engorged mosquitoes over this period by amplifying specific human microsatellite markers in mosquito blood meals and human cheek swabs. Using a multinomial model assuming a saturating relationship (power), we found that, relative to other residents of a home, an individual''s likelihood of being bitten in the home was directly proportional to time spent in their home and body surface area (p<0.05). A linear function fit the relationship equally well (ΔAIC<1).

Conclusions/Significance

Our results indicate that larger people and those who spend more time at home are more likely to receive Ae. aegypti bites in their homes than other household residents. These findings are consistent with the idea that measurable characteristics of individuals can inform predictions of the extent to which different people will be bitten. This has implications for an improved understanding of heterogeneity in different people''s contributions to pathogen transmission, and enhanced interventions that include the people and places that contribute most to pathogen amplification and spread.     

Shifting Patterns of Aedes aegypti Fine Scale Spatial Clustering in Iquitos,Peru

Background

Empiric evidence shows that Aedes aegypti abundance is spatially heterogeneous and that some areas and larval habitats produce more mosquitoes than others. There is a knowledge gap, however, with regards to the temporal persistence of such Ae. aegypti abundance hotspots. In this study, we used a longitudinal entomologic dataset from the city of Iquitos, Peru, to (1) quantify the spatial clustering patterns of adult Ae. aegypti and pupae counts per house, (2) determine overlap between clusters, (3) quantify the temporal stability of clusters over nine entomologic surveys spaced four months apart, and (4) quantify the extent of clustering at the household and neighborhood levels.

Methodologies/Principal Findings

Data from 13,662 household entomological visits performed in two Iquitos neighborhoods differing in Ae. aegypti abundance and dengue virus transmission was analyzed using global and local spatial statistics. The location and extent of Ae. aegypti pupae and adult hotspots (i.e., small groups of houses with significantly [p<0.05] high mosquito abundance) were calculated for each of the 9 entomologic surveys. The extent of clustering was used to quantify the probability of finding spatially correlated populations. Our analyses indicate that Ae. aegypti distribution was highly focal (most clusters do not extend beyond 30 meters) and that hotspots of high vector abundance were common on every survey date, but they were temporally unstable over the period of study.

Conclusions/Significance

Our findings have implications for understanding Ae. aegypti distribution and for the design of surveillance and control activities relying on household-level data. In settings like Iquitos, where there is a relatively low percentage of Ae. aegypti in permanent water-holding containers, identifying and targeting key premises will be significantly challenged by shifting hotspots of Ae. aegypti infestation. Focusing efforts in large geographic areas with historically high levels of transmission may be more effective than targeting Ae. aegypti hotspots.     

Genetic Diversity of Brazilian Aedes aegypti: Patterns following an Eradication Program

Background

Aedes aegypti is the most important vector of dengue fever in Brazil, where severe epidemics have recently taken place. Ae. aegypti in Brazil was the subject of an intense eradication program in the 1940s and 50s to control yellow fever. Brazil was the largest country declared free of this mosquito by the Pan-American Health Organization in 1958. Soon after relaxation of this program, Ae. aegypti reappeared in this country, and by the early 1980s dengue fever had been reported. The aim of this study is to analyze the present-day genetic patterns of Ae. aegypti populations in Brazil.

Methodology/Principal Findings

We studied the genetic variation in samples of 11 widely spread populations of Ae. aegypti in Brazil based on 12 well-established microsatellite loci. Our principal finding is that present-day Brazilian Ae. aegypti populations form two distinct groups, one in the northwest and one in the southeast of the country. These two groups have genetic affinities to northern South American countries and the Caribbean, respectively. This is consistent with what has been reported for other genetic markers such as mitochondrial DNA and allele frequencies at the insecticide resistance gene, kdr.

Conclusions/Significance

We conclude that the genetic patterns in present day populations of Ae. aegypti in Brazil are more consistent with a complete eradication of the species in the recent past followed by re-colonization, rather than the alternative possibility of expansion from residual pockets of refugia. At least two colonizations are likely to have taken place, one from northern South American countries (e.g., Venezuela) that founded the northwestern group, and one from the Caribbean that founded the southeastern group. The proposed source areas were never declared free of Ae. aegypti.     

Trisomy in Aedes aegypti.

A trisomic (2n=6+1) pupa of the yellow fever mosquito Aedes aegypti has been found. The trisomy involved chromosome 3 which is intermediate in size between 1 and 2. The extra chromosome formed a univalent or a trivalent during meiosis.     

Ammonia metabolism in Aedes aegypti

We investigated the mechanisms by which Aedes aegypti mosquitoes are able to metabolize ammonia. When females were given access to solutions containing NH(4)Cl or to a blood meal, hemolymph glutamine and proline concentrations increased markedly, indicating that ammonium/ammonia can be removed from the body through the synthesis of these two amino acids. The importance of glutamine synthetase was shown when an inhibitor of the enzyme was added to the meal causing the glutamine concentration in hemolymph to decrease significantly, while the proline concentration increased dramatically. Unexpectedly, we found an important role for glutamate synthase. When mosquitoes were fed azaserine, an inhibitor of glutamate synthase, the glutamine concentration increased and the proline concentration decreased significantly. This confirms the presence of glutamate synthase in mosquitoes and suggests that this enzyme contributes to the production of glutamate for proline synthesis. Several key enzymes related to ammonium/ammonia metabolism showed activity in homogenates of mosquito fat body and midgut. The mosquito genes encoding glutamate dehydrogenase, glutamine synthetase, glutamate synthase, pyrroline-5-carboxylate synthase were cloned and sequenced. The mRNA expression patterns of these genes were examined by a real-time RT-PCR in fat body and midgut. The results show that female mosquitoes have evolved efficient mechanisms to detoxify large loads of ammonium/ammonia.     

Population Genetics of Plasmodium vivax in the Peruvian Amazon

Background

Characterizing the parasite dynamics and population structure provides useful information to understand the dynamic of transmission and to better target control interventions. Despite considerable efforts for its control, vivax malaria remains a major health problem in Peru. In this study, we have explored the population genetics of Plasmodium vivax isolates from Iquitos, the main city in the Peruvian Amazon, and 25 neighbouring peri-urban as well as rural villages along the Iquitos-Nauta Road.

Methodology/ Results

From April to December 2008, 292 P. vivax isolates were collected and successfully genotyped using 14 neutral microsatellites. Analysis of the molecular data revealed a similar proportion of monoclonal and polyclonal infections in urban areas, while in rural areas monoclonal infections were predominant (p = 0.002). Multiplicity of infection was higher in urban (MOI = 1.5–2) compared to rural areas (MOI = 1) (p = 0.003). The level of genetic diversity was similar in all areas (He = 0.66–0.76, p = 0.32) though genetic differentiation between areas was substantial (PHI_PT = 0.17, p<0.0001). Principal coordinate analysis showed a marked differentiation between parasites from urban and rural areas. Linkage disequilibrium was detected in all the areas (IAs = 0.08–0.49, for all p<0.0001). Gene flow among the areas was stablished through Bayesian analysis of migration models. Recent bottleneck events were detected in 4 areas and a recent parasite expansion in one of the isolated areas. In total, 87 unique haplotypes grouped in 2 or 3 genetic clusters described a sub-structured parasite population.

Conclusion/Significance

Our study shows a sub-structured parasite population with clonal propagation, with most of its components recently affected by bottleneck events. Iquitos city is the main source of parasite spreading for all the peripheral study areas. The routes of transmission and gene flow and the reduction of the parasite population described are important from the public health perspective as well for the formulation of future control policies.     

Forest Canopy Gap Distributions in the Southern Peruvian Amazon

Canopy gaps express the time-integrated effects of tree failure and mortality as well as regrowth and succession in tropical forests. Quantifying the size and spatial distribution of canopy gaps is requisite to modeling forest functional processes ranging from carbon fluxes to species interactions and biological diversity. Using high-resolution airborne Light Detection and Ranging (LiDAR), we mapped and analyzed 5,877,937 static canopy gaps throughout 125,581 ha of lowland Amazonian forest in Peru. Our LiDAR sampling covered a wide range of forest physiognomies across contrasting geologic and topographic conditions, and on depositional floodplain and erosional terra firme substrates. We used the scaling exponent of the Zeta distribution (λ) as a metric to quantify and compare the negative relationship between canopy gap frequency and size across sites. Despite variable canopy height and forest type, values of λ were highly conservative (λ _mean  = 1.83, s  = 0.09), and little variation was observed regionally among geologic substrates and forest types, or at the landscape level comparing depositional-floodplain and erosional terra firme landscapes. λ-values less than 2.0 indicate that these forests are subjected to large gaps that reset carbon stocks when they occur. Consistency of λ-values strongly suggests similarity in the mechanisms of canopy failure across a diverse array of lowland forests in southwestern Amazonia.     

Midgut bacterial dynamics in Aedes aegypti

In vector mosquitoes, the presence of midgut bacteria may affect the ability to transmit pathogens. We have used a laboratory colony of Aedes aegypti as a model for bacterial interspecies competition and show that after a blood meal, the number of species (culturable on Luria-Bertani agar) that coexist in the midgut is low and that about 40% of the females do not harbor any cultivable bacteria. We isolated species belonging to the genera Bacillus, Elizabethkingia, Enterococcus, Klebsiella, Pantoea, Serratia, and Sphingomonas, and we also determined their growth rates, antibiotic resistance, and ex vivo inhibition of each other. To investigate the possible existence of coadaptation between midgut bacteria and their host, we fed Ae.?aegypti cohorts with gut bacteria from human, a frog, and two mosquito species and followed the bacterial population growth over time. The dynamics of the different species suggests coadaptation between host and bacteria, and interestingly, we found that Pantoea stewartii isolated from Ae.?aegypti survive better in Ae.?aegypti as compared to P.?stewartii isolated from the malaria mosquito Anopheles gambiae.     

The ultrastructure of the Aedes aegypti heart

Comparative structural analyses of the heart and associated tissues in 4th instar larvae (L4), pupae and adults of Aedes aegypti were undertaken using a combination of microscopy techniques. The Ae. aegypti heart consists of cardiomyocytes arranged in a helical fashion, and it is physically associated with intersegmental groups of pericardial cells (PCs) and the alary muscles (AMs). Ramifications commonly present in AMs are more developed in adults than in the immature stages. Pericardial cells absorb and store extracellular components as shown by the uptake of carmine dye fed in larval diet. We also observed that carmine stained inclusions corresponding to electron-dense structures resembling lysosomes that were more abundant and prominent in pupae, suggestive of increase of waste accumulation during pupation. The results presented here expand on previously known aspects of the mosquito heart and describe for the first time comparative aspects of the morphology of the heart in different developmental stages.     

Population structure of the mosquito Aedes aegypti (Stegomyia aegypti) in Pakistan

Eleven microsatellite markers were used to determine the genetic population structure and spread of Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) in Pakistan using mosquitoes collected from 13 different cities. There is a single genetic cluster of Ae. aegypti in Pakistan with a pattern of isolation by distance within the population. The low level of isolation by distance suggests the long‐range passive dispersal of this mosquito, which may be facilitated by the tyre trade in Pakistan. A decrease in genetic diversity from south to north suggests a recent spread of this mosquito from Karachi. A strong negative correlation between genetic distance and the quality of road connections shows that populations in cities connected by better road networks are less differentiated, which suggests the human‐aided passive dispersal of Ae. aegypti in Pakistan. Dispersal on a large spatial scale may facilitate the strategy of introducing transgenic Ae. aegypti or intracellular bacteria such as Wolbachia to control the spread of dengue disease in Pakistan, but it also emphasizes the need for simple measures to control container breeding sites.     

Strategies for genetic conservation of trees in the Peruvian Amazon

Forestry practices and high rates of land clearance for agriculture are causing genetic erosion of valuable tree species in the Peruvian Amazon, thereby endangering the economic sustainability of rural communities and limiting Peru's opportunities for the development of new timber and non-timber forest products. The potential utility and limitations of six low-input interventions to help forestall further genetic erosion in the region are discussed, with a focus on local community involvement. Improved agroforestry systems may help reduce deforestation by increasing farm productivity, although methods to increase the currently low adoption rate of these technologies need to be developed. Use of strategic tree domestication techniques can also improve farm productivity and prevent inadvertent genetic drift and inbreeding associated with traditional domestication practices, although to have a major impact, current programs need to be extended across the region. Woodlot forestry could supplant selective extraction of timber and offers an attractive opportunity for poverty alleviation if appropriate credit and land tenure policies can be developed. However, it may also result in increased deforestation if activities on public land cannot be controlled. The implementation of improved seed collection systems and simple seed transfer guidelines would help to reduce the collection of seed of poor quality and low genetic diversity, and avoid maladapted plantings, although such programs are difficult to monitor and seed costs may increase. Strategic identification and design of in situ conservation areas would help to ensure the viability of conserved populations, but requires the forfeiture of significant revenue from timber concessions.