Liaisons dangereuses: cross-border gene flow and dispersal of insecticide resistance-associated genes in the mosquito Aedes aegypti from Brazil and French Guiana.

BACKGROUND: In recent years, South America has suffered the burden of continuous high impact outbreaks of dengue, chikungunya and Zika. Aedes aegypti is the main mosquito vector of these arboviruses and its control is the only solution to reduce transmission. OBJECTIVES: In order to improve vector control it is essential to study mosquito population genetics in order to better estimate the population structures and the geneflow among them. METHODS: We have analysed microsatellites and knockdown resistance (kdr) mutations from a trans-border region in Amazonia between the state of Amapá (Brazil) and French Guiana (overseas territory of France), to provide further knowledge on these issues. These two countries have followed distinct vector control policies since last century. For population genetic analyses we evaluated variability in 13 well-established microsatellites loci in Ae. aegypti from French Guiana (Saint Georges and Cayenne) and Brazil (Oiapoque and Macapá). The occurrence and frequency of kdr mutations in these same populations were accessed by TaqMan genotype assays for the sites 1016 (Val/Ile) and 1534 (Phe/Cys). FINDINGS: We have detected high levels of gene flow between the closest cross-border samples of Saint-Georges and Oiapoque. These results suggest one common origin of re-colonisation for the populations of French Guiana and Oiapoque in Brazil, and a different source for Macapá, more similar to the other northern Brazilian populations. Genotyping of the kdr mutations revealed distinct patterns for Cayenne and Macapá associated with their different insecticide use history, and an admixture zone between these two patterns in Saint Georges and Oiapoque, in accordance with population genetic results. MAIN CONCLUSIONS: The present study highlights the need for regional-local vector surveillance and transnational collaboration between neighboring countries to assess the impact of implemented vector control strategies, promote timely actions and develop preparedness plans.

Plasmodium falciparum Genetic Diversity in Continental Equatorial Guinea before and after Introduction of Artemisinin-Based Combination Therapy.

Efforts to control malaria may affect malaria parasite genetic variability and drug resistance, the latter of which is associated with genetic events that promote mechanisms to escape drug action. The worldwide spread of drug resistance has been a major obstacle to controlling Plasmodium falciparum malaria, and thus the study of the origin and spread of associated mutations may provide some insights into the prevention of its emergence. This study reports an analysis of P. falciparum genetic diversity, focusing on antimalarial resistance-associated molecular markers in two socioeconomically different villages in mainland Equatorial Guinea. The present study took place 8 years after a previous one, allowing the analysis of results before and after the introduction of an artemisinin-based combination therapy (ACT), i.e., artesunate plus amodiaquine. Genetic diversity was assessed by analysis of the Pfmsp2 gene and neutral microsatellite loci. Pfdhps and Pfdhfr alleles associated with sulfadoxine-pyrimethamine (SP) resistance and flanking microsatellite loci were investigated, and the prevalences of drug resistance-associated point mutations of the Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genes were estimated. Further, to monitor the use of ACT, we provide the baseline prevalences of K13 propeller mutations and Pfmdr1 copy numbers. After 8 years, noticeable differences occurred in the distribution of genotypes conferring resistance to chloroquine and SP, and the spread of mutated genotypes differed according to the setting. Regarding artemisinin resistance, although mutations reported as being linked to artemisinin resistance were not present at the time, several single nucleotide polymorphisms (SNPs) were observed in the K13 gene, suggesting that closer monitoring should be maintained to prevent the possible spread of artemisinin resistance in Africa.

Seasonal genetic partitioning in the neotropical malaria vector, Anopheles darlingi.

BACKGROUND: Anopheles darlingi is the main malaria mosquito vector in the Amazonia region. In spite of being considered a riverine, forest-dwelling species, this mosquito is becoming more abundant in peri-urban areas, increasing malaria risk. This has been associated with human-driven environmental changes such as deforestation. METHODS: Microsatellites were used to characterize A. darlingi from seven localities along the Madeira River, Rondônia (Brazil), collected in the early and late periods of the rainy season. RESULTS: Two genetically distinct subpopulations were detected: one (subpopulation A) was associated with the late rainfall period and seems to be ecologically closer to the typical forest A. darlingi; the other (subpopulation B) was associated with the early rainfall period and is probably more adapted to drier conditions by exploiting permanent anthropogenic breeding sites. Results suggest also a pattern of asymmetric introgression, with more subpopulation A alleles introgressed into subpopulation B. Both subpopulations (and admixed mosquitoes) presented similar malaria infection rates, highlighting the potential for perennial malaria transmission in the region. CONCLUSIONS: The co-occurrence of two genetically distinct subpopulations of A. darlingi adapted to different periods of rainfall may promote a more perennial transmission of malaria throughout the year. These findings, in a context of strong environmental impact due to deforestation and dam construction, have serious implications for malaria epidemiology and control in the Amazonian region.

Genetic diversity and signatures of selection of drug resistance in Plasmodium populations from both human and mosquito hosts in continental Equatorial Guinea.

BACKGROUND: In Plasmodium, the high level of genetic diversity and the interactions established by co-infecting parasite populations within the same host may be a source of selection on pathogen virulence and drug resistance. As different patterns have already been described in humans and mosquitoes, parasite diversity and population structure should be studied in both hosts to properly assess their effects on infection and transmission dynamics. This study aimed to characterize the circulating populations of Plasmodium spp and Plasmodium falciparum from a combined set of human blood and mosquito samples gathered in mainland Equatorial Guinea. Further, the origin and evolution of anti-malarial resistance in this area, where malaria remains a major public health problem were traced. METHODS: Plasmodium species infecting humans and mosquitoes were identified by nested-PCR of chelex-extracted DNA from dried blood spot samples and mosquitoes. Analysis of Pfmsp2 gene, anti-malarial-resistance associated genes, Pfdhps, Pfdhfr, Pfcrt and Pfmdr1, neutral microsatellites (STR) loci and Pfdhfr and Pfdhps flanking STR was undertaken to evaluate P. falciparum diversity. RESULTS: Prevalence of infection remains high in mainland Equatorial Guinea. No differences in parasite formula or significant genetic differentiation were seen in the parasite populations in both human and mosquito samples. Point mutations in all genes associated with anti-malarial resistance were highly prevalent. A high prevalence was observed for the Pfdhfr triple mutant in particular, associated with pyrimethamine resistance.Analysis of Pfdhps and Pfdhfr flanking STR revealed a decrease in the genetic diversity. This finding along with multiple independent introductions of Pfdhps mutant haplotypes suggest a soft selective sweep and an increased differentiation at Pfdhfr flanking microsatellites hints a model of positive directional selection for this gene. CONCLUSIONS: Chloroquine is no longer recommended for malaria treatment in Equatorial Guinea but sulphadoxine-pyrimethamine (SP) remains in use in combination with artesunate and is the only drug recommended in preventive chemotherapy in pregnancy. The high prevalence of point mutations in Pfdhfr and Pfdhps points to the danger of an eventual reduction in the efficacy of SP combined therapy in P. falciparum populations in Equatorial Guinea and to the essential continuous monitoring of these two genes.

Aedes aegypti on Madeira Island (Portugal): genetic variation of a recently introduced dengue vector.

The increasing population of Aedes aegypti mosquitoes on Madeira Island (Portugal) resulted in the first autochthonous dengue outbreak, which occurred in October 2012. Our study establishes the first genetic evaluation based on the mitochondrial DNA (mtDNA) genes [cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4)] and knockdown resistance (kdr) mutations exploring the colonisation history and the genetic diversity of this insular vector population. We included mosquito populations from Brazil and Venezuela in the analysis as putative geographic sources. The Ae. aegypti population from Madeira showed extremely low mtDNA genetic variability, with a single haplotype for COI and ND4. We also detected the presence of two important kdr mutations and the quasi-fixation of one of these mutations (F1534C). These results are consistent with a unique recent founder event that occurred on the island of Ae. aegypti mosquitoes that carry kdr mutations associated with insecticide resistance. Finally, we also report the presence of the F1534C kdr mutation in the Brazil and Venezuela populations. To our knowledge, this is the first time this mutation has been found in South American Ae. aegypti mosquitoes. Given the present risk of Ae. aegypti re-invading continental Europe from Madeira and the recent dengue outbreaks on the island, this information is important to plan surveillance and control measures.

Genetic and phenotypic variation of the malaria vector Anopheles atroparvus in southern Europe.

BACKGROUND: There is a growing concern that global climate change will affect the potential for pathogen transmission by insect species that are vectors of human diseases. One of these species is the former European malaria vector, Anopheles atroparvus. Levels of population differentiation of An. atroparvus from southern Europe were characterized as a first attempt to elucidate patterns of population structure of this former malaria vector. Results are discussed in light of a hypothetical situation of re-establishment of malaria transmission. METHODS: Genetic and phenotypic variation was analysed in nine mosquito samples collected from five European countries, using eight microsatellite loci and geometric morphometrics on 21 wing landmarks. RESULTS: Levels of genetic diversity were comparable to those reported for tropical malaria vectors. Low levels of genetic (0.004

Tracing the origins and signatures of selection of antifolate resistance in island populations of Plasmodium falciparum.

BACKGROUND: Resistance of the malaria parasite Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) has evolved worldwide. In the archipelago of São Tomé and Principe (STP), West Africa, although SP resistance is highly prevalent the drug is still in use in particular circumstances. To address the evolutionary origins of SP resistance in these islands, we genotyped point mutations at P. falciparum dhfr and dhps genes and analysed microsatellites flanking those genes. METHODS: Blood samples were collected in July and December 2004 in three localities of São Tomé Island and one in Principe Island. Species-specific nested-PCR was used to identify P. falciparum infected samples. Subsequently, SNPs at the dhfr and dhps genes were identified through PCR-RFLP. Isolates were also analysed for three microsatellite loci flanking the dhfr gene, three loci flanking dhps and four loci located at putative neutral genomic regions. RESULTS: An increase of resistance-associated mutations at dhfr and dhps was observed, in particular for the dhfr/dhps quintuple mutant, associated with clinical SP failure. Analysis of flanking microsatellites suggests multiple independent introductions for dhfr and dhps mutant haplotypes, possibly from West Africa. A reduced genetic diversity and increased differentiation at flanking microsatellites when compared to neutral loci is consistent with a selective sweep for resistant alleles at both loci. CONCLUSIONS: This study provides additional evidence for the crucial role of gene flow and drug selective pressures in the rapid spread of SP resistance in P. falciparum populations, from only a few mutation events giving rise to resistance-associated mutants. It also highlights the importance of human migration in the spread of drug resistant malaria parasites, as the distance between the islands and mainland is not consistent with mosquito-mediated parasite dispersal.

Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region, Portugal.

BACKGROUND: Culex pipiens L. is the most widespread mosquito vector in temperate regions. This species consists of two forms, denoted molestus and pipiens, that exhibit important behavioural and physiological differences. The evolutionary relationships and taxonomic status of these forms remain unclear. In northern European latitudes molestus and pipiens populations occupy different habitats (underground vs. aboveground), a separation that most likely promotes genetic isolation between forms. However, the same does not hold in southern Europe where both forms occur aboveground in sympatry. In these southern habitats, the extent of hybridisation and its impact on the extent of genetic divergence between forms under sympatric conditions has not been clarified. For this purpose, we have used phenotypic and genetic data to characterise Cx. pipiens collected aboveground in Portugal. Our aims were to determine levels of genetic differentiation and the degree of hybridisation between forms occurring in sympatry, and to relate these with both evolutionary and epidemiological tenets of this biological group. RESULTS: Autogeny and stenogamy was evaluated in the F1 progeny of 145 individual Cx. pipiens females. Bayesian clustering analysis based on the genotypes of 13 microsatellites revealed two distinct genetic clusters that were highly correlated with the alternative traits that define pipiens and molestus. Admixture analysis yielded hybrid rate estimates of 8-10%. Higher proportions of admixture were observed in pipiens individuals suggesting that more molestus genes are being introgressed into the pipiens form than the opposite. CONCLUSION: Both physiological/behavioural and genetic data provide evidence for the sympatric occurrence of molestus and pipiens forms of Cx. pipiens in the study area. In spite of the significant genetic differentiation between forms, hybridisation occurs at considerable levels. The observed pattern of asymmetric introgression probably relates to the different mating strategies adopted by each form. Furthermore, the differential introgression of molestus genes into the pipiens form may induce a more opportunistic biting behaviour in the latter thus potentiating its capacity to act as a bridge-vector for the transmission of arboviral infections.

Population structure of a cave-dwelling bat, Miniopterus schreibersii: does it reflect history and social organization?

Many colonial bat species make regional migrations, and the consequent gene flow may eliminate geographic genetic structure resulting from history of colonization. In this study, we verified that history and social organization have detectable impacts on the genetic structure of Miniopterus schreibersii, a cave-dwelling bat with high female philopatry. After studying all known nursing colonies in Portugal, we concluded that there is a significant geographic structure and that the overall pattern is similar for mitochondrial and nuclear DNA. Both pairwise Phi(ST) and F(ST) were significantly correlated with geographical distance, suggesting that isolation by distance is relevant for both mitochondrial and nuclear markers. However, structuring of mitochondrial DNA was much more marked than that of nuclear DNA, a consequence of the strong female philopatry and a bias for male-mediated gene flow. Wintering colonies were more genetically diverse than nursing colonies because the former receive individuals from distinct breeding populations. Haplotype diversity of the northern colonies, the more recent according to population expansion analyses, is only about half of that of the central and southern colonies. This is most likely a consequence of the colonization history of M. schreibersii, which presumably expanded northward from the south of the Iberian Peninsula or North Africa after the last glacial age.

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.

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.

Genetic population structure of Anopheles gambiae in Equatorial Guinea.

BACKGROUND: Patterns of genetic structure among mosquito vector populations in islands have received particular attention as these are considered potentially suitable sites for experimental trials on transgenic-based malaria control strategies. In this study, levels of genetic differentiation have been estimated between populations of Anopheles gambiae s.s. from the islands of Bioko and Annobón, and from continental Equatorial Guinea (EG) and Gabon. METHODS: Genotyping of 11 microsatellite loci located in chromosome 3 was performed in three island samples (two in Bioko and one in Annobón) and three mainland samples (two in EG and one in Gabon). Four samples belonged to the M molecular form and two to the S-form. Microsatellite data was used to estimate genetic diversity parameters, perform demographic equilibrium tests and analyse population differentiation. RESULTS: High levels of genetic differentiation were found between the more geographically remote island of Annobón and the continent, contrasting with the shallow differentiation between Bioko island, closest to mainland, and continental localities. In Bioko, differentiation between M and S forms was higher than that observed between island and mainland samples of the same molecular form. CONCLUSION: The observed patterns of population structure seem to be governed by the presence of both physical (the ocean) and biological (the M-S form discontinuity) barriers to gene flow. The significant degree of genetic isolation between M and S forms detected by microsatellite loci located outside the "genomic islands" of speciation identified in A. gambiae s.s. further supports the hypothesis of on-going incipient speciation within this species. The implications of these findings regarding vector control strategies are discussed.

Genetic diversity and population structure of Plasmodium falciparum over space and time in an African archipelago.

The archipelago of São Tomé and Principe (STP), West Africa, has suffered the heavy burden of malaria since the 16th century. Until the last decade, when after a successful control program STP has become a low transmission country and one of the few nations with decreases of more than 90% in malaria admission and death rates. We carried out a longitudinal study to determine the genetic structure of STP parasite populations over time and space. Twelve microsatellite loci were genotyped in Plasmodium falciparum samples from two islands collected in 1997, 2000 and 2004. Analysis was performed on proportions of mixed genotype infections, allelic diversity, population differentiation, effective population size and bottleneck effects. We have found high levels of genetic diversity and minimal inter-population genetic differentiation typical of African continental regions with intense and stable malaria transmission. We detected significant differences between the years, with special emphasis for 1997 that showed the highest proportion of samples infected with P. falciparum and the highest mean number of haplotypes per isolate. This study establishes a comprehensive genetic data baseline of a pre-intervention scenario for future studies; taking into account the most recent and successful control intervention on the territory.

Molecular evolution and population genetics of a Gram-negative binding protein gene in the malaria vector Anopheles gambiae (sensu lato).

BACKGROUND: Clarifying the role of the innate immune system of the malaria vector Anopheles gambiae is a potential way to block the development of the Plasmodium parasites. Pathogen recognition is the first step of innate immune response, where pattern recognition proteins like GNBPs play a central role. RESULTS: We analysed 70 sequences of the protein coding gene GNBPB2 from two species, Anopheles gambiae (s.s.) and An. coluzzii, collected in six African countries. We detected 135 segregating sites defining 63 distinct haplotypes and 30 proteins. Mean nucleotide diversity (π) was 0.014 for both species. We found no significant genetic differentiation between species, but a significant positive correlation between genetic differentiation and geographical distance among populations. CONCLUSIONS: Species status seems to contribute less for the molecular differentiation in GNBPB2 than geographical region in the African continent (West and East). Purifying selection was found to be the most common form of selection, as in many other immunity-related genes. Diversifying selection may be also operating in the GNBPB2 gene.

Population diversity of Theileria annulata in Portugal.

The tick-borne protozoan parasite Theileria annulata causes tropical theileriosis, a severe disease of cattle that occurs across the Mediterranean littoral, the Middle East and Southern Asia. In the Mediterranean region, the disease has long been perceived as being a constraint to livestock production in North Africa and Turkey but was believed to have minimal impact in Southern European countries. It has recently been demonstrated that in Southern Portugal the prevalence of T. annulata is approximately 30%. While the population genetics of the parasite and the multiplicity of infection in the bovine host have been studied in a number of countries, no information is currently available on the composition of the parasite population in Southern Europe or its relationship to populations in bordering regions. A parasite genotyping system, based on micro- and mini-satellite amplification, was used to perform genetic analysis of T. annulata populations from T. annulata infected cattle in twelve farms in Southern Portugal. A diversity of genotypes and a high multiplicity of infection were found, suggesting that the parasite possesses a panmictic population in this region. In comparison with genotypes found in Tunisia and Turkey, parasites from Portugal form a genetically distinct group and show lower genetic diversity.

Liaisons dangereuses: cross-border gene flow and dispersal of insecticide resistance-associated genes in the mosquito Aedes aegypti from Brazil and French Guiana

BACKGROUND In recent years, South America has suffered the burden of continuous high impact outbreaks of dengue, chikungunya and Zika. Aedes aegypti is the main mosquito vector of these arboviruses and its control is the only solution to reduce transmission. OBJECTIVES In order to improve vector control it is essential to study mosquito population genetics in order to better estimate the population structures and the geneflow among them. METHODS We have analysed microsatellites and knockdown resistance (kdr) mutations from a trans-border region in Amazonia between the state of Amapá (Brazil) and French Guiana (overseas territory of France), to provide further knowledge on these issues. These two countries have followed distinct vector control policies since last century. For population genetic analyses we evaluated variability in 13 well-established microsatellites loci in Ae. aegypti from French Guiana (Saint Georges and Cayenne) and Brazil (Oiapoque and Macapá). The occurrence and frequency of kdr mutations in these same populations were accessed by TaqMan genotype assays for the sites 1016 (Val/Ile) and 1534 (Phe/Cys). FINDINGS We have detected high levels of gene flow between the closest cross-border samples of Saint-Georges and Oiapoque. These results suggest one common origin of re-colonisation for the populations of French Guiana and Oiapoque in Brazil, and a different source for Macapá, more similar to the other northern Brazilian populations. Genotyping of the kdr mutations revealed distinct patterns for Cayenne and Macapá associated with their different insecticide use history, and an admixture zone between these two patterns in Saint Georges and Oiapoque, in accordance with population genetic results. MAIN CONCLUSIONS The present study highlights the need for regional-local vector surveillance and transnational collaboration between neighboring countries to assess the impact of implemented vector control strategies, promote timely actions and develop preparedness plans.

Effects of hemoglobin variants on hemoglobin a1c values measured using a high-performance liquid chromatography method.

Hemoglobin A1c (HbA1c) is routinely used to monitor long-term glycemic control and for diagnosing diabetes mellitus. However, hemoglobin (Hb) gene variants/modifications can affect the accuracy of some methods. The potential effect of Hb variants on HbA1c measurements was investigated using a high-performance liquid chromatography (HPLC) method compared with an immunoturbimetric assay. Fasting plasma glucose (FPG) and HbA1c levels were measured in 42 371 blood samples. Samples producing abnormal chromatograms were further analyzed to characterize any Hb variants. Fructosamine levels were determined in place of HbA1c levels when unstable Hb variants were identified. Abnormal HPLC chromatograms were obtained for 160 of 42 371 samples. In 26 samples HbS was identified and HbA1c results correlated with FPG. In the remaining 134 samples HbD, Hb Louisville, Hb Las Palmas, Hb N-Baltimore, or Hb Porto Alegre were identified and HbA1c did not correlate with FPG. These samples were retested using an immunoturbidimetric assay and the majority of results were accurate; only 3 (with the unstable Hb Louisville trait) gave aberrant HbA1c results. Hb variants can affect determination of HbA1c levels with some methods. Laboratories should be aware of Hb variants occurring locally and choose an appropriate HbA1c testing method.

New insights into the population structure of Anopheles gambiae s.s. in the Gulf of Guinea Islands revealed by Herves transposable elements.

Transposable elements (TEs) are mobile portions of DNA that are able to replicate and spread in the genome of many organisms. TEs can be used as a means to insert transgenes in insects, being stably inherited throughout generations. Anopheles gambiae is the main vector of human malaria in Sub-Saharan Africa. Given the extraordinary burden this disease imposes, the mosquito became a choice target for genetic control approaches with the purpose of reducing malaria transmission. In this study, we investigated the abundance and distribution of Herves TE in An. gambiae s.s. from Cameroon and four islands in the Gulf of Guinea, in order to determine their genetic structure. We have detected a population subdivision between Equatorial Guinea islands and the islands of São Tomé, Príncipe and mainland. This partitioning associates more with political rather than geographic boundaries, possibly reflecting different mainland source populations colonizing the islands.