Screening of natural Wolbachia infection in mosquitoes (Diptera: Culicidae) from the Cape Verde islands.

BACKGROUND: Wolbachia pipientis is an endosymbiont bacterium that induces cytoplasmic incompatibility and inhibits arboviral replication in mosquitoes. This study aimed to assess Wolbachia prevalence and genetic diversity in different mosquito species from Cape Verde. METHODS: Mosquitoes were collected on six islands of Cape Verde and identified to species using morphological keys and PCR-based assays. Wolbachia was detected by amplifying a fragment of the surface protein gene (wsp). Multilocus sequence typing (MLST) was performed with five housekeeping genes (coxA, gatB, ftsZ, hcpA, and fbpA) and the wsp hypervariable region (HVR) for strain identification. Identification of wPip groups (wPip-I to wPip-V) was performed using PCR-restriction fragment length polymorphism (RFLP) assay on the ankyrin domain gene pk1. RESULTS: Nine mosquito species were collected, including the major vectors Aedes aegypti, Anopheles arabiensis, Culex pipiens sensu stricto, and Culex quinquefasciatus. Wolbachia was only detected in Cx. pipiens s.s. (100% prevalence), Cx. quinquefasciatus (98.3%), Cx. pipiens/quinquefasciatus hybrids (100%), and Culex tigripes (100%). Based on the results of MLST and wsp hypervariable region typing, Wolbachia from the Cx. pipiens complex was assigned to sequence type 9, wPip clade, and supergroup B. PCR/RFLP analysis revealed three wPip groups in Cape Verde, namely wPip-II, wPip-III, and wPip-IV. wPip-IV was the most prevalent, while wPip-II and wPip-III were found only on Maio and Fogo islands. Wolbachia detected in Cx. tigripes belongs to supergroup B, with no attributed MLST profile, indicating a new strain of Wolbachia in this mosquito species. CONCLUSIONS: A high prevalence and diversity of Wolbachia was found in species from the Cx. pipiens complex. This diversity may be related to the mosquito's colonization history on the Cape Verde islands. To the best of our knowledge, this is the first study to detect Wolbachia in Cx. tigripes, which may provide an additional opportunity for biocontrol initiatives.

Wide and increasing suitability for Aedes albopictus in Europe is congruent across distribution models.

The Asian tiger mosquito (Aedes albopictus), a vector of dengue, Zika and other diseases, was introduced in Europe in the 1970s, where it is still widening its range. Spurred by public health concerns, several studies have delivered predictions of the current and future distribution of the species for this region, often with differing results. We provide the first joint analysis of these predictions, to identify consensus hotspots of high and low suitability, as well as areas with high uncertainty. The analysis focused on current and future climate conditions and was carried out for the whole of Europe and for 65 major urban areas. High consensus on current suitability was found for the northwest of the Iberian Peninsula, southern France, Italy and the coastline between the western Balkans and Greece. Most models also agree on a substantial future expansion of suitable areas into northern and eastern Europe. About 83% of urban areas are expected to become suitable in the future, in contrast with ~ 49% nowadays. Our findings show that previous research is congruent in identifying wide suitable areas for Aedes albopictus across Europe and in the need to effectively account for climate change in managing and preventing its future spread.

A Case for Systematic Quality Management in Mosquito Control Programmes in Europe.

The recent spread of invasive mosquito species, such as Aedes albopictus and the seasonal sporadic transmission of autochthonous cases of arboviral diseases (e.g., dengue, chikungunya, Zika) in temperate areas, such as Europe and North America, highlight the importance of effective mosquito-control interventions to reduce not only nuisance, but also major threats for public health. Local, regional, and even national mosquito control programs have been established in many countries and are executed on a seasonal basis by either public or private bodies. In order for these interventions to be worthwhile, funding authorities should ensure that mosquito control is (a) planned by competent scientific institutions addressing the local demands, (b) executed following the plan that is based on recommended and effective methods and strategies, (c) monitored regularly by checking the efficacy of the implemented actions, (d) evaluated against the set of targets, and (e) regularly improved according to the results of the monitoring. Adherence to these conditions can only be assured if a formal quality management system is adopted and enforced that ensures the transparency of effectiveness of the control operation. The current paper aims at defining the two components of this quality management system, quality assurance and quality control for mosquito control programs with special emphasis on Europe, but applicable over temperate areas.

The V410L knockdown resistance mutation occurs in island and continental populations of Aedes aegypti in West and Central Africa.

The extensive use of insecticides for vector control has led to the development of insecticide resistance in Aedes aegypti populations on a global scale, which has significantly compromised control actions. Insecticide resistance, and its underlying mechanisms, has been investigated in several countries, mostly in South American and Asian countries. In Africa, however, studies reporting insecticide resistance are rare and data on resistance mechanisms, notably knockdown resistance (kdr) mutations, is scarce. In this study, the recently described V410L kdr mutation is reported for the first time in old world Ae. aegypti populations, namely from Angola and Madeira island. Two additional kdr mutations, V1016I and F1534C, are also reported for the first time in populations from Angola and Cape Verde. Significant associations with the resistance phenotype were found for both V410L and V1016I individually as well as for tri-locus genotypes in the Angolan population. However, no association was found in Madeira island, probably due to the presence of a complex pattern of multiple insecticide resistance mechanisms in the local Ae. aegypti population. These results suggest that populations carrying the same kdr mutations may respond differently to the same insecticide, stressing the need for complementary studies when assessing the impact of kdr resistance mechanisms in the outcome of insecticide-based control strategies.

Practical management plan for invasive mosquito species in Europe: I. Asian tiger mosquito (Aedes albopictus).

Aedes albopictus, also known as the "Asian Tiger Mosquito", is an invasive mosquito species to Europe causing high concern in public health due to its severe nuisance and its vectorial capacity for pathogens such as dengue, chikungunya, yellow fever and Zika. Consequently, the responsible authorities implement management activities to reduce its population density, possibly to below noxious and epidemiological thresholds. In urban areas, these aims are difficult to achieve because of the species' ability to develop in a wide range of artificial breeding sites, mainly private properties. This document (Management Plan) has been structured to serve as a comprehensive practical and technical guide for stakeholders in organizing the vector control activities in the best possible way. The current plan includes coordinated actions such as standardized control measures and quality control activities, monitoring protocols, activities for stakeholders and local communities, and an emergency vector control plan to reduce the risk of an epidemic.

What happens when we modify mosquitoes for disease prevention? A systematic review.

The release of modified mosquitoes to suppress/replace vectors constitutes a promising tool for vector control and disease prevention. Evidence regarding these innovative modification techniques is scarce and disperse. This work conducted a systematic review, gathering and analysing research articles from PubMed and Biblioteca Virtual em Saúde databases whose results report efficacy and non-target effects of using modified insects for disease prevention, until 2016. More than 1500 publications were screened and 349 were analysed. Only 12/3.4% articles reported field-based evidence and 41/11.7% covered modification strategies' post-release efficacy. Variability in the effective results (90/25.7%) questioned its reproducibility in different settings. We also found publications reporting reversal outcomes 38/10.9%, (e.g. post-release increase of vector population). Ecological effects were also reported, such as horizontal transfer events (54/15.5%), and worsening pathogenesis induced by natural wolbachia (10/2.9%). Present work revealed promising outcomes of modifying strategies. However, it also revealed a need for field-based evidence mainly regarding epidemiologic and long-term impact. It pointed out some eventual irreversible and important effects that must not be ignored when considering open-field releases, and that may constitute constraints to generate the missing field evidence. Present work constitutes a baseline of knowledge, offering also a methodological approach that may facilitate future updates.

Phylogeography and invasion history of Aedes aegypti, the Dengue and Zika mosquito vector in Cape Verde islands (West Africa).

Aedes-borne arboviruses have spread globally with outbreaks of vast impact on human populations and health systems. The West African archipelago of Cape Verde had its first outbreak of Dengue in 2009, at the time the largest recorded in Africa, and was one of the few African countries affected by the Zika virus epidemic. Aedes aegypti was the mosquito vector involved in both outbreaks. We performed a phylogeographic and population genetics study of A. aegypti in Cape Verde in order to infer the geographic origin and evolutionary history of this mosquito. These results are discussed with respect to the implications for vector control and prevention of future outbreaks. Mosquitoes captured before and after the Dengue outbreak on the islands of Santiago, Brava, and Fogo were analyzed with two mitochondrial genes COI and ND4, 14 microsatellite loci and five kdr mutations. Genetic variability was comparable to other African populations. Our results suggest that A. aegypti invaded Cape Verde at the beginning of the Holocene from West Africa. Given the historic importance of Cape Verde in the transatlantic trade of the 16th-17th centuries, a possible contribution to the genetic pool of the founding populations in the New World cannot be fully discarded. However, contemporary gene flow with the Americas is likely to be infrequent. No kdr mutations associated with pyrethroid resistance were detected. The implications for vector control and prevention of future outbreaks are discussed.

An evaluation of efficacy of the auto-dissemination technique as a tool for Aedes aegypti control in Madeira, Portugal.

BACKGROUND: The frequency and intensity of arboviral epidemics is steadily increasing and posing an intractable public health burden. Current vector control methods are proving ineffectual and despite progress in the development of high technology approaches, there is an urgent need for the development of tools for immediate implementation. Several studies suggest that the auto-dissemination of pyriproxyfen (PPF) is a promising new approach to larviciding although there is little detail on the conditions under which it is optimally effective. Here, we evaluate the efficacy of the approach in urban and rural sites in Madeira, Portugal. RESULTS: Auto-dissemination of PPF through adapted Biogents Sentinel traps (BGSTs) resulted in a modest but consistent impact on both juvenile and adult mosquito populations, but with considerable spatial heterogeneity. This heterogeneity was related to the distance from the BGST dissemination station as well as the local density of adult mosquitoes. There was evidence that the impact of PPF was cumulative over time both locally and with gradual spatial expansion. CONCLUSIONS: The density of adult mosquitoes and the spatial distribution of dissemination devices are key factors in mediating efficacy. In addition, urban topography may affect the efficiency of auto-dissemination by impeding adult mosquito dispersal. Further studies in a range of urban landscapes are necessary to guide optimal strategies for the implementation of this potentially efficacious and cost-effective approach to larviciding.

Origin and expansion of the mosquito Aedes aegypti in Madeira Island (Portugal).

Historically known as the yellow fever mosquito, Aedes aegypti invaded Madeira Island in 2005 and was the vector of the island's first dengue outbreak in 2012. We have studied genetic variation at 16 microsatellites and two mitochondrial DNA genes in temporal samples of Madeira Island, in order to assess the origin of the invasion and the population structure of this mosquito vector. Our results indicated at least two independent colonization events occurred on the island, both having a South American source population. In both scenarios, Venezuela was the most probable origin of these introductions, a result that is in accordance with the socioeconomic relations between this country and Madeira Island. Once introduced, Ae. aegypti has rapidly expanded along the southern coast of the island and reached a maximum effective population size (Ne) in 2012, coincident with the dengue epidemic. After the outbreak, there was a 10-fold reduction in Ne estimates, possibly reflecting the impact of community-based vector control measures implemented during the outbreak. These findings have implications for mosquito surveillance not only for Madeira Island, but also for other European regions where Aedes mosquitoes are expanding.

Population structure of a vector of human diseases: Aedes aegypti in its ancestral range, Africa.

Aedes aegypti, the major vector of dengue, yellow fever, chikungunya, and Zika viruses, remains of great medical and public health concern. There is little doubt that the ancestral home of the species is Africa. This mosquito invaded the New World 400-500 years ago and later, Asia. However, little is known about the genetic structure and history of Ae. aegypti across Africa, as well as the possible origin(s) of the New World invasion. Here, we use ~17,000 genome-wide single nucleotide polymorphisms (SNPs) to characterize a heretofore undocumented complex picture of this mosquito across its ancestral range in Africa. We find signatures of human-assisted migrations, connectivity across long distances in sylvan populations, and of local admixture between domestic and sylvan populations. Finally, through a phylogenetic analysis combined with the genetic structure analyses, we suggest West Africa and especially Angola as the source of the New World's invasion, a scenario that fits well with the historic record of 16th-century slave trade between Africa and Americas.

From the Laboratory to the Field: Updating Capacity Building in Medical Entomology.

Training and innovation in the field of medical entomology are essential to mitigate the burden of vector-borne diseases globally. However, there is a shortage of medical entomologists worldwide, and there are large discrepancies in capacity building in this field. In this article, we discuss the current situation, what is needed from the medical entomologist of today, and how we can bridge this gap.

Massive introgression drives species radiation at the range limit of Anopheles gambiae.

Impacts of introgressive hybridisation may range from genomic erosion and species collapse to rapid adaptation and speciation but opportunities to study these dynamics are rare. We investigated the extent, causes and consequences of a hybrid zone between Anopheles coluzzii and Anopheles gambiae in Guinea-Bissau, where high hybridisation rates appear to be stable at least since the 1990s. Anopheles gambiae was genetically partitioned into inland and coastal subpopulations, separated by a central region dominated by A. coluzzii. Surprisingly, whole genome sequencing revealed that the coastal region harbours a hybrid form characterised by an A. gambiae-like sex chromosome and massive introgression of A. coluzzii autosomal alleles. Local selection on chromosomal inversions may play a role in this process, suggesting potential for spatiotemporal stability of the coastal hybrid form and providing resilience against introgression of medically-important loci and traits, found to be more prevalent in inland A. gambiae.

Global genetic diversity of Aedes aegypti.

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co-occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub-Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans-Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.

Zika Virus, a New Threat for Europe?

BACKGROUND: Since its emergence in 2007 in Micronesia and Polynesia, the arthropod-borne flavivirus Zika virus (ZIKV) has spread in the Americas and the Caribbean, following first detection in Brazil in May 2015. The risk of ZIKV emergence in Europe increases as imported cases are repeatedly reported. Together with chikungunya virus (CHIKV) and dengue virus (DENV), ZIKV is transmitted by Aedes mosquitoes. Any countries where these mosquitoes are present could be potential sites for future ZIKV outbreak. We assessed the vector competence of European Aedes mosquitoes (Aedes aegypti and Aedes albopictus) for the currently circulating Asian genotype of ZIKV. METHODOLOGY/PRINCIPAL FINDINGS: Two populations of Ae. aegypti from the island of Madeira (Funchal and Paul do Mar) and two populations of Ae. albopictus from France (Nice and Bar-sur-Loup) were challenged with an Asian genotype of ZIKV isolated from a patient in April 2014 in New Caledonia. Fully engorged mosquitoes were then maintained in insectary conditions (28°±1°C, 16h:8h light:dark cycle and 80% humidity). 16-24 mosquitoes from each population were examined at 3, 6, 9 and 14 days post-infection to estimate the infection rate, disseminated infection rate and transmission efficiency. Based on these experimental infections, we demonstrated that Ae. albopictus from France were not very susceptible to ZIKV. CONCLUSIONS/SIGNIFICANCE: In combination with the restricted distribution of European Ae. albopictus, our results on vector competence corroborate the low risk for ZIKV to expand into most parts of Europe with the possible exception of the warmest regions bordering the Mediterranean coastline.

Limited genomic divergence between intraspecific forms of Culex pipiens under different ecological pressures.

BACKGROUND: Divergent selection can be a major driver of ecological speciation. In insects of medical importance, understanding the speciation process is both of academic interest and public health importance. In the West Nile virus vector Culex pipiens, intraspecific pipiens and molestus forms vary in ecological and physiological traits. Populations of each form appear to share recent common ancestry but patterns of genetic differentiation across the genome remain unknown. Here, we undertook an AFLP genome scan on samples collected from both sympatric and allopatric populations from Europe and the USA to quantify the extent of genomic differentiation between the two forms. RESULTS: The forms were clearly differentiated but each exhibited major population sub-structuring between continents. Divergence between pipiens and molestus forms from USA was higher than in both inter- and intra-continental comparisons with European samples. The proportion of outlier loci between pipiens and molestus (≈3 %) was low but consistent in both continents, and similar to those observed between sibling species of other mosquito species which exhibit contemporary gene flow. Only two of the outlier loci were shared between inter-form comparisons made within Europe and USA. CONCLUSION: This study supports the molestus and pipiens status as distinct evolutionary entities with low genomic divergence. The low number of shared divergent loci between continents suggests a relatively limited number of genomic regions determining key typological traits likely to be driving incipient speciation and/or adaptation of molestus to anthropogenic habitats.

Approaches to passive mosquito surveillance in the EU.

The recent emergence in Europe of invasive mosquitoes and mosquito-borne disease associated with both invasive and native mosquito species has prompted intensified mosquito vector research in most European countries. Central to the efforts are mosquito monitoring and surveillance activities in order to assess the current species occurrence, distribution and, when possible, abundance, in order to permit the early detection of invasive species and the spread of competent vectors. As active mosquito collection, e.g. by trapping adults, dipping preimaginal developmental stages or ovitrapping, is usually cost-, time- and labour-intensive and can cover only small parts of a country, passive data collection approaches are gradually being integrated into monitoring programmes. Thus, scientists in several EU member states have recently initiated programmes for mosquito data collection and analysis that make use of sources other than targeted mosquito collection. While some of them extract mosquito distribution data from zoological databases established in other contexts, community-based approaches built upon the recognition, reporting, collection and submission of mosquito specimens by citizens are becoming more and more popular and increasingly support scientific research. Based on such reports and submissions, new populations, extended or new distribution areas and temporal activity patterns of invasive and native mosquito species were found. In all cases, extensive media work and communication with the participating individuals or groups was fundamental for success. The presented projects demonstrate that passive approaches are powerful tools to survey the mosquito fauna in order to supplement active mosquito surveillance strategies and render them more focused. Their ability to continuously produce biological data permits the early recognition of changes in the mosquito fauna that may have an impact on biting nuisance and the risk of pathogen transmission associated with mosquitoes. International coordination to explore synergies and increase efficiency of passive surveillance programmes across borders needs to be established.

Dengue fever in Europe: could there be an epidemic in the future?

Dengue virus (DENV) is the arbovirus with the widest impact on human health. Although its dispersal is partially conditioned by environmental constraints that limit the distribution of its main vector (Aedes aegypti), DENV has been spreading geographically in recent times, but mostly afflicting tropical and subtropical regions. With no prophylactic vaccine or specific therapeutics available, vector control remains the best alternative to restrain its circulation. Moreover, the establishment of thriving vector populations in peri urban environments brings humans and viruses together, opening the possibility for the occurrence of unexpected outbreaks. Europe is no exception: such was the case of Madeira in 2012. In addition to its impact on the health of the local population, health services, and economy, this outbreak revealed how difficult it may be to control the circulation of pathogenic arboviruses, especially taking into consideration that Europe is already partially colonized by another DENV vector, Aedes albopictus.

First report of an exophilic Anopheles arabiensis population in Bissau City, Guinea-Bissau: recent introduction or sampling bias?

BACKGROUND: The malaria vector Anopheles arabiensis exhibits greater behavioural and ecological plasticity than the other major vectors of the Anopheles gambiae complex, which presents challenges for major control methods. This study reports for the first time the presence of An. arabiensis in Antula, a suburb of Bissau city, the capital of Guinea Bissau, where high levels of hybridization between Anopheles coluzzii and An. gambiae have been reported. Given that previous surveys in the area, based on indoor collections, did not sample An. arabiensis, the possibility of a recently introduced exophilic population was investigated. METHODS: Larval and adult mosquito collections were carried out in Antula at the end of the rainy season of 2010. Anopheles gambiae species composition, determined by rDNA-IGS and SINE200X6.1 markers, was compared with four previously collected samples dating back to 1993. Analysis of ten microsatellites was used to estimate levels of genetic diversity, relatedness and to investigate demographic stability. RESULTS: Anopheles arabiensis comprised 54.0% of larvae and 25.6% of adults collected in 2010, but was absent in all previous collections, a highly unlikely observation by chance if the population was stable. This species had the lowest levels of genetic diversity, highest relatedness and, along with An. gambiae, exhibited evidence of a recent population expansion. CONCLUSIONS: Results point to the presence of a previously undetected outdoor population of An. arabiensis in Antula, which appears to have expanded recently, highlighting the importance of complementing indoor-based mosquito collections with sampling methods targeting outdoor adults and immature stages for a more complete assessment of mosquito biodiversity. A change in temporal dynamics in the species complex composition was also detected. Coupled with previous evidence of asymmetric introgression from An. coluzzii to An. gambiae, this suggests that the study area may be subject to ecological changes with a potential impact on both the genetics of these species and on malaria transmission.

Macroclimate determines the global range limit of Aedes aegypti.

Aedes aegypti is the main vector of dengue and a number of other diseases worldwide. Because of the domestic nature of this mosquito, the relative importance of macroclimate in shaping its distribution has been a controversial issue. We have captured here the worldwide macroclimatic conditions occupied by A. aegypti in the last century. We assessed the ability of this information to predict the species' observed distribution using supra-continental spatially-uncorrelated data. We further projected the distribution of the colonized climates in the near future (2010-2039) under two climate-change scenarios. Our results indicate that the macroclimate is largely responsible for setting the maximum range limit of A. aegypti worldwide and that in the near future, relatively wide areas beyond this limit will receive macroclimates previously occupied by the species. By comparing our projections, with those from a previous model based strictly on species-climate relationships (i.e., excluding human influence), we also found support for the hypothesis that much of the species' range in temperate and subtropical regions is being sustained by artificial environments. Altogether, these findings suggest that, if the domestic environments commonly exploited by this species are available in the newly suitable areas, its distribution may expand considerably in the near future.

Feeding patterns of molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in a region of high hybridization.

BACKGROUND: Two biological forms of the mosquito Culex pipiens s.s., denoted pipiens and molestus, display behavioural differences that may affect their role as vectors of arboviruses. In this study, the feeding patterns of molestus and pipiens forms were investigated in Comporta (Portugal), where high levels of inter-form admixture have been recorded. METHODS: Indoor and outdoor mosquito collections were performed in the summer of 2010. Collected Cx. pipiens s.l. females were molecularly identified to species and form by PCR and genotyped for six microsatellites. The source of the blood meal in post-fed females was determined by ELISA and mitochondrial DNA sequencing. RESULTS: The distribution of the forms differed according to the collection method. The molestus form was present only in indoor collections, whereas pipiens and admixed individuals were sampled both indoors and outdoors. In both forms, over 90% of blood meals were made on avian hosts. These included blood meals taken from Passeriformes (Passer domesticus and Turdus merula) by females caught resting inside domestic shelters. CONCLUSION: Genetic structure and blood meal analyses suggest the presence of a bird biting molestus population in the study area. Both forms were found to rest indoors, mainly in avian shelters, but at least a proportion of females of the pipiens form may bite outdoors in sylvan habitats and then search for anthropogenic resting sites to complete their gonotrophic cycle. This behaviour may potentiate the accidental transmission of arboviruses to humans in the region.