André Barretto Bruno Wilke

Paratransgenesis: a promising new strategy for mosquito vector control

The three main mosquito genera, Anopheles, Aedes and Culex, transmit respectively malaria, dengue and lymphatic filariasis. Current mosquito control strategies have proved unsuccessful, and there still is a substantial number of morbidity and mortality from these diseases. Genetic control methods have now arisen as promising alternative strategies, based on two approaches: the replacement of a vector population by disease-refractory mosquitoes and the release of mosquitoes carrying a lethal gene to suppress target populations. However, substantial hurdles and limitations need to be overcome if these methods are to be used successfully, the most significant being that a transgenic mosquito strain is required for every target species, making genetically modified mosquito strategies inviable when there are multiple vector mosquitoes in the same area. Genetically modified bacteria capable of colonizing a wide range of mosquito species may be a solution to this problem and another option for the control of these diseases. In the paratransgenic approach, symbiotic bacteria are genetically modified and reintroduced in mosquitoes, where they express effector molecules. For this approach to be used in practice, however, requires a better understanding of mosquito microbiota and that symbiotic bacteria and effector molecules be identified. Paratransgenesis could prove very useful in mosquito species that are inherently difficult to transform or in sibling species complexes. In this approach, a genetic modified bacteria can act by: (a) causing pathogenic effects in the host; (b) interfering with the host’s reproduction; (c) reducing the vector’s competence; and (d) interfering with oogenesis and embryogenesis. It is a much more flexible and adaptable approach than the use of genetically modified mosquitoes because effector molecules and symbiotic bacteria can be replaced if they do not achieve the desired result. Paratransgenesis may therefore become an important integrated pest management tool for mosquito control.

Genetic Control of Mosquitoes: population suppression strategies

Over the last two decades, morbidity and mortality from malaria and dengue fever among other pathogens are an increasing Public Health problem. The increase in the geographic distribution of vectors is accompanied by the emergence of viruses and diseases in new areas. There are insufficient specific therapeutic drugs available and there are no reliable vaccines for malaria or dengue, although some progress has been achieved, there is still a long way between its development and actual field use. Most mosquito control measures have failed to achieve their goals, mostly because of the mosquitos great reproductive capacity and genomic flexibility. Chemical control is increasingly restricted due to potential human toxicity, mortality in no target organisms, insecticide resistance, and other environmental impacts. Other strategies for mosquito control are desperately needed. The Sterile Insect Technique (SIT) is a species-specific and environmentally benign method for insect population suppression, it is based on mass rearing, radiation mediated sterilization, and release of a large number of male insects. Releasing of Insects carrying a dominant lethal gene (RIDL) offers a solution to many of the drawbacks of traditional SIT that have limited its application in mosquitoes while maintaining its environmentally friendly and species-specific utility. The self-limiting nature of sterile mosquitoes tends to make the issues related to field use of these somewhat less challenging than for self-spreading systems characteristic of population replacement strategies. They also are closer to field use, so might be appropriate to consider first. The prospect of genetic control methods against mosquito vectored human diseases is rapidly becoming a reality, many decisions will need to be made on a national, regional and international level regarding the biosafety, social, cultural and ethical aspects of the use and deployment of these vector control methods.

Population genetics of neotropical Culex quinquefasciatus (Diptera: Culicidae)

BackgroundCulex quinquefasciatus mosquitoes can be found in almost every major city of Brazil and are vectors of filariasis and several arboviruses. Microsatellite markers have been widely used to uncover the genetic structure of various groups of insect populations. The aim of this study was to glimpse the genetic structure of Cx. quinquefasciatus in Brazil.MethodsNine populations were sampled across Brazil (one of them from a laboratory colony - COL) and another one from Argentina and process regarding the variability of six microsatellite loci.ResultsThe analyzed loci revealed moderate population genetic structure (mean Fst = 0.12). Dendrograms of genetic distances evidenced two major population clusters, respectively corresponding to the northern and southern populations. The hybrid population Cx. pipiens/quinquefasciatus (from La Plata, Argentina) and the colony population fell outside the major clusters. Those clusters were substructured and there was a significant correlation between genetic and geographic distances and environmental variables (r = 0.51; p > 0.001 and r = 0.46; p > 0.004).ConclusionsMultilocus cluster Bayesian analysis confirmed that populations are mutually distinct, and the set of results point to genetic differences among populations. The presumable low gene flow among them may be due to the large geographic distances (>1000 km) and to the environmental heterogeneity of the sampled areas. The genetic structure observed in this study may lead to the best understanding of Cx. quinquefasciatus demographical diversity as well as their genetic variations patterns in Brazil so far unknown.

Morphometric Wing Characters as a Tool for Mosquito Identification

Mosquitoes are responsible for the transmission of important infectious diseases, causing millions of deaths every year and endangering approximately 3 billion people around the world. As such, precise identification of mosquito species is crucial for an understanding of epidemiological patterns of disease transmission. Currently, the most common method of mosquito identification relies on morphological taxonomic keys, which do not always distinguish cryptic species. However, wing geometric morphometrics is a promising tool for the identification of vector mosquitoes, sibling and cryptic species included. This study therefore sought to accurately identify mosquito species from the three most epidemiologically important mosquito genera using wing morphometrics. Twelve mosquito species from three epidemiologically important genera (Aedes, Anopheles and Culex) were collected and identified by taxonomic keys. Next, the right wing of each adult female mosquito was removed and photographed, and the coordinates of eighteen digitized landmarks at the intersections of wing veins were collected. The allometric influence was assessed, and canonical variate analysis and thin-plate splines were used for species identification. Cross-validated reclassification tests were performed for each individual, and a Neighbor Joining tree was constructed to illustrate species segregation patterns. The analyses were carried out and the graphs plotted with TpsUtil 1.29, TpsRelw 1.39, MorphoJ 1.02 and Past 2.17c. Canonical variate analysis for Aedes, Anopheles and Culex genera showed three clear clusters in morphospace, correctly distinguishing the three mosquito genera, and pairwise cross-validated reclassification resulted in at least 99% accuracy; subgenera were also identified correctly with a mean accuracy of 96%, and in 88 of the 132 possible comparisons, species were identified with 100% accuracy after the data was subjected to reclassification. Our results showed that Aedes, Culex and Anopheles were correctly distinguished by wing shape. For the lower hierarchical levels (subgenera and species), wing geometric morphometrics was also efficient, resulting in high reclassification scores.

Mosquito Faunal Survey In a Central Park of the City of São Paulo, Brazil

Abstract A total of 2,582 specimens of mosquitoes of 16 taxonomic categories grouped into 5 genera (Aedes, Culex, Mansonia, Toxorhynchites, and Wyeomyia) were collected in a central park of São Paulo City, Brazil. It is crucial to include such an area in official entomological surveillance programs since this park has all the epidemiological characteristics needed to maintain an enzootic cycle of arboviruses.

Population Genetic Structure of Aedes fluviatilis (Diptera: Culicidae)

Although Aedes fluviatilis is an anthropophilic mosquito found abundantly in urban environments, its biology, epidemiological potential and genetic characteristics are poorly understood. Climate change and urbanization processes that result in environmental modifications benefit certain anthropophilic mosquito species such as Ae. fluviatilis, greatly increasing their abundance in urban areas. To gain a better understanding of whether urbanization processes modulate the genetic structure of this species in the city of São Paulo, we used eight microsatellite loci to genetically characterize Ae. fluviatilis populations collected in nine urban parks in the city of São Paulo. Our results show that there is high gene flow among the populations of this species, heterozygosity deficiency and low genetic structure and that the species may have undergone a recent population expansion. There are two main hypotheses to explain these findings: (i) Ae. fluviatilis populations have undergone a population expansion as a result of urbanization; and (ii) as urbanization of the city of São Paulo occurred recently and was quite intense, the structuring of these populations cannot be observed yet, apart from in the populations of Ibirapuera and Piqueri parks, where the first signs of structuring have appeared. We believe that the expansion found in Ae. fluviatilis populations is probably correlated with the unplanned urbanization of the city of São Paulo, which transformed green areas into urbanized areas, as well as the increasing population density in the city.

Mosquito populations dynamics associated with climate variations.

Mosquitoes are responsible for the transmission of numerous serious pathogens. Members of the Aedes and Culex genera, which include many important vectors of mosquito-borne diseases, are highly invasive and adapted to man-made environments. They are spread around the world involuntarily by humans and are highly adapted to urbanized environments, where they are exposed to climate-related abundance drivers. We investigated Culicidae fauna in two urban parks in the city of São Paulo to analyze the correlations between climatic variables and the population dynamics of mosquitoes in these urban areas. Mosquitoes were collected monthly over one year, and sampling sufficiency was evaluated after morphological identification of the specimens. The average monthly temperature and accumulated rainfall for the collection month and previous month were used to explain climate-related abundance drivers for the six most abundant species (Aedes aegypti, Aedes albopictus, Aedes fluviatilis, Aedes scapularis, Culex nigripalpus and Culex quinquefasciatus) and then analyzed using generalized linear statistical models and the Akaike Information Criteria corrected for small samples (AICc). The strength of evidence in favor of each model was evaluated using Akaike weights, and the explanatory model power was measured by McFaddens Pseudo-R2. Associations between climate and mosquito abundance were found in both parks, indicating that predictive models based on climate variables can provide important information on mosquito population dynamics. We also found that this association is species-dependent. Urbanization processes increase the abundance of a few mosquito species that are well adapted to man-made environments and some of which are important vectors of pathogens. Predictive models for abundance based on climate variables may help elucidate the population dynamics of urban mosquitoes and their impact on the risk of disease transmission, allowing better predictive scenarios to be developed and supporting the implementation of vector mosquito control strategies.

Microsatellite loci cross-species transferability in Aedes fluviatilis (Diptera:Culicidae): a cost-effective approach for population genetics studies

BackgroundAedes fluviatilis is a neotropical mosquito species thought to be a potential vector of Yellow Fever viruses and can be infected with Plasmodium gallinaceum in laboratory. A better understanding of its genetic structure is very important to understand its epidemiologic potential and how it is responding to urbanization. The objective of this study was to survey the transferability of microsatellites loci developed for other Aedes to Ae. fluviatilis.FindingsWe tested in Ae. fluviatilis 40 pairs of primers known to flank microsatellite regions in Aedes aegypti, Aedes albopictus and Aedes caspius, and found eight loci that amplified consistently. The number of alleles per locus ranged from 2 to 15, and the expected heterozygosity ranged from 0.09 to 0.85.ConclusionsWe found that several microsatellite primers successfully transferred to Ae. fluviatilis. This finding opens avenues for cost-effective optimization of high-resolution population genetic tools.

Microgeographic population structuring of Aedes aegypti (Diptera: Culicidae)

Aedes aegypti is one of the species most favored by changes in the environment caused by urbanization. Its abundance increases rapidly in the face of such changes, increasing the risk of disease transmission. Previous studies have shown that mosquito species that have adapted to anthropogenic environmental changes benefit from urbanization and undergo population expansion. In light of this, we used microsatellite markers to explore how urbanization processes may be modulating Ae. aegypti populations collected from three areas with different levels of urbanization in the city of São Paulo, Brazil. Specimens were collected at eleven sites in three areas with different degrees of urbanization in the city of São Paulo: conserved, intermediate and urbanized. Ten microsatellite loci were used to characterize the populations from these areas genetically. Our findings suggest that as urbanized areas grow and the human population density in these areas increases, Ae. aegypti populations undergo a major population expansion, which can probably be attributed to the species’ adaptability to anthropogenic environmental changes. Our findings reveal a robust association between, on the one hand, urbanization processes and densification of the human population and, on the other, Ae. aegypti population structure patterns and population expansion. This indicates that this species benefits from anthropogenic effects, which are intensified by migration of the human population from rural to urban areas, increasing the risk of epidemics and disease transmission to an ever-increasing number of people.

Wing variation in Culex nigripalpus (Diptera: Culicidae) in urban parks

BackgroundCulex nigripalpus has a wide geographical distribution and is found in North and South America. Females are considered primary vectors for several arboviruses, including Saint Louis encephalitis virus, Venezuelan equine encephalitis virus and Eastern equine encephalitis virus, as well as a potential vector of West Nile virus. In view of the epidemiological importance of this mosquito and its high abundance, this study sought to investigate wing variation in Cx. nigripalpus populations from urban parks in the city of São Paulo, Brazil.MethodsFemale mosquitoes were collected in seven urban parks in the city of São Paulo between 2011 and 2013. Eighteen landmark coordinates from the right wing of each female mosquito were digitized, and the dissimilarities between populations were assessed by canonical variate analysis and cross-validated reclassification and by constructing a Neighbor-Joining (NJ) tree based on Mahalanobis distances. The centroid size was calculated to determine mean wing size in each population.ResultsCanonical variate analysis based on fixed landmarks of the wing revealed a pattern of segregation between urban and sylvatic Cx. nigripalpus, a similar result to that revealed by the NJ tree topology, in which the population from Shangrilá Park segregated into a distinct branch separate from the other more urban populations.ConclusionEnvironmental heterogeneity may be affecting the wing shape variation of Cx. nigripalpus populations.