Patricia Salgueiro | Universidade Nova de Lisboa (original) (raw)

articles by Patricia Salgueiro

Historically known as the yellow fever mosquito, Aedes aegypti invaded Madeira Island in 2005 and... more 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.

Antimicrob Agents Chemother. 2017 Jan; 61(1): e02556-15. , 2017

Efforts to control malaria may affect malaria parasite genetic variability and drug resistance, t... more 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. fal-ciparum 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.

Background Clarifying the role of the innate immune system of the malaria vector Anopheles gambi... more 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.

The archipelago of São Tomé and Principe (STP), West Africa, has suffered the heavy burden of mal... more 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.

The tick-borne protozoan parasite Theileria annulata causes tropical theileriosis, a severe disea... more 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.

BioMed Research International, 2016

Background: Anopheles darlingi is the main malaria mosquito vector in the Amazonia region. In spi... more 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.

The increasing population of Aedes aegypti mosquitoes on Madeira Island (Portugal) resulted in th... more 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.

BMC Evolutionary Biology, 2009

Background: Culex pipiens L. is the most widespread mosquito vector in temperate regions. This sp... more 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.

Bulletin of Entomological Research, 2011

The 'paddy paradox', the occurrence of large populations of vectors but low amounts of malaria tr... more The 'paddy paradox', the occurrence of large populations of vectors but low amounts of malaria transmission where irrigated rice is grown, was investigated in a village in Ghana where M form Anopheles gambiae are common. Peridomestic and indoor host-seeking mosquitoes were collected in tent traps and light traps over 21 consecutive nights at the start of the rainy season in June 2009 when the population increased exponentially from less than 100 per night to over 1000. Infection rates in the overall mosquito population were 0.3% and in the estimated parous population were 1.9%. Numbers of An. gambiae in the tent trap peaked between midnight and 02:40 am. The majority of insects were taking their first blood meal, as virgins or shortly after mating. More than expected were collected in the light trap during a rainstorm at the start of the rains but overall numbers were not affected. Fewer than expected were collected after a subsequent storm. Recruitment to the adult population decreased over the following days. It is hypothesised that the 'paddy paradox' is due to young pre-gravid insects dispersing more widely than gravid ones, not necessarily to low survival in the mosquito.

Background Resistance of the malaria parasite Plasmodium falciparum to sulfadoxine-pyrimethamin... more 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.

ABSTRACT: Patterns of genetic structure among mosquito vector populations in islands have receive... more ABSTRACT: 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. 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. 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. 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.

Conservation Genetics, 2008

The Azorean bat (Nyctalus azoreum) is endemic to the Azores archipelago and is listed as endanger... more The Azorean bat (Nyctalus azoreum) is endemic to the Azores archipelago and is listed as endangered due to its reduced and fragmented distribution range. We assessed genetic diversity at eight microsatellite loci in 280 individuals from 14 locations throughout six islands. Overall, we found that the Azorean bat populations are not genetically impoverished. Indeed, the number of alleles per locus ranged from 8 to 10 and the observed heterozygosity ranged from 0.77 in Terceira to 0.83 in Faial. The highest genetic diversity and level of private alleles was observed in S. Miguel, the largest island, and the closest to the mainland. Private alleles occurred at all islands except in Graciosa. Global and pairwise F ST among islands were all statistically significant, suggesting restricted gene flow. These results, together with those of factorial correspondence analysis, Bayesian clustering method, and individual assignment tests, corroborate the conclusions of a previous mtDNA based study, providing strong support for the existence of two major subpopulations: one includes all islands of the Central Group and the other corresponds to S. Miguel. Gene flow between them is very limited, suggesting that management plans should avoid translocations between these subpopulations.

Genetica, 2007

We used three mitochondrial DNA fragments with different substitution rates (ND1, Cyt b and the C... more We used three mitochondrial DNA fragments with different substitution rates (ND1, Cyt b and the CR) to infer phylogenetic relationships among six species of the genus Nyctalus, and compare levels of genetic divergence between the insular, vulnerable Nyctalus azoreum and its continental counterpart to assess the origins of the Azorean bat. The larger species found throughout the Palaearctic region (N. lasiopterus, N. aviator and N. noctula) share a unique chromosome formula (2n = 42) and form a monophyletic clade in our reconstructions. Nyctalus plancyi (= velutinus), a Chinese taxon with 2n = 36 chromosomes, is sometimes included in N. noctula, but is genetically very divergent from the latter and deserves full species status. All Cyt b and CR haplotypes of N. azoreum are closely related and only found in the Azores archipelago, but when compared to continental sequences of N. leisleri, levels of mtDNA divergence are unusually low for mammalian species. This contrasts with the high level of differentiation that N. azoreum has attained in its morphology, ecology, and echolocation calls, suggesting a recent split followed by fast evolutionary change. The molecular data suggest that N. azoreum originated from a European population of N. leisleri, and that the colonisation of the Azores occurred at the end of the Pleistocene. The Madeiran populations of N. leisleri also appear to have a European origin, whereas those of the Canary Islands probably came from North Africa. In spite of its recent origin and low genetic divergence, the Azorean bat is well differentiated and consequently represents a unique evolutionary unit with great conservation value.

Journal of Heredity, 2009

Many colonial bat species make regional migrations, and the consequent gene flow may eliminate ge... more 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 U 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.

Journal of Medical Entomology, 2008

In the malaria vector Anopheles gambiae Giles sensu stricto, two molecular forms denoted M and S ... more In the malaria vector Anopheles gambiae Giles sensu stricto, two molecular forms denoted M and S are considered units of incipient speciation within this species. Very low hybrid frequencies and signiÞcant genetic differentiation have been found in sympatric M-and S-form populations. We studied the molecular form composition and the degree of genetic differentiation at 15 microsatellites in two samples of An. gambiae collected in two consecutive years from Bissau, Guinea Bissau. High frequencies of M/S hybrids (19 Ð24%) were found in this area. Coincidently, very low levels of genetic differentiation were detected between forms when analysis involved microsatellites mapped at chromosome-3 (mean F st , 0.000 Ð 0.002). The single exception was the X-linked AGXH678, for which high differentiation was measured (F st , 0.158 Ð 0.301). This locus maps near the centromere of chromosome X, a low recombination region in which selection is likely to promote divergence between M and S forms. These results strongly suggest that the degree of isolation between M and S forms, considered the units of incipient speciation within An. gambiae, is not homogenous throughout the species distribution range.

PLoS ONE, 2013

Transposable elements (TEs) are mobile portions of DNA that are able to replicate and spread in t... more 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. Citation: Salgueiro P, Moreno M, Simard F, O'Brochta D, Pinto J (2013) New Insights into the Population Structure of Anopheles gambiae s.s. in the Gulf of Guinea Islands Revealed by Herves Transposable Elements. PLoS ONE 8(4): e62964.

Malaria journal, 2011

There is a growing concern that global climate change will affect the potential for pathogen tran... more 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. 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. Levels of genetic diversity were comparable to those reported for tropical malaria vectors. Low levels of genetic (0.004 <FST <0.086) and phenotypic differentiation were detected among An. atroparvus populations spanning over 3,000 km distance. Genetic differen...

Malaria Journal, 2013

Background: In Plasmodium, the high level of genetic diversity and the interactions established b... more 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.

Antimicrob Agents Chemother. 2017 Jan; 61(1): e02556-15. , 2017

Background Clarifying the role of the innate immune system of the malaria vector Anopheles gambi... more Background

BioMed Research International, 2016

BMC Evolutionary Biology, 2009

Bulletin of Entomological Research, 2011

Background Resistance of the malaria parasite Plasmodium falciparum to sulfadoxine-pyrimethamin... more Background

Conservation Genetics, 2008

Genetica, 2007

Journal of Heredity, 2009

Journal of Medical Entomology, 2008

PLoS ONE, 2013

Malaria journal, 2011

Malaria Journal, 2013

II Workshop sobre clima e recursos naturais nos países de língua portuguesa: livro de actas, 2010

Resumo No contexto actual de alterações climáticas e ambientais, a preocupação com doenças (re) e... more Resumo No contexto actual de alterações climáticas e ambientais, a preocupação com doenças (re) emergentes torna-se cada vez maior. Em particular, tem-se vindo a dar mais atenção a doenças transmitidas por insectos vectores, das quais se destaca a malária, pelo enorme impacto que tem na população humana. Num cenário potencial de ressurgimento da malária na Europa, a taxa de transmissão de parasitas Plasmodium importados será certamente afectada por padrões de estrutura e de conectividade entre as populações do vector. E este é o tema central do presente trabalho aplicado ao antigo mosquito vector de malária em Portugal, Anopheles atroparvus. Assim, foram genotipados 10 marcadores moleculares (microssatélites) em mosquitos colhidos em 10 locais de Portugal entre 1993 e 2008. Os dados obtidos sugerem um fluxo genético generalizado no país, e uma correlação positiva entre a distância geográfica e a diferenciação genética das populações de A. atroparvus. Estimativas de tamanho efectivo populacional (Ne) baseadas em amostras temporais variaram entre 1990 e 12197 indivíduos por geração. Foram detectados indícios de contracção populacional em quase todas as amostras. Estes resultados podem ser ainda consequência das intensas medidas anti-vector tomadas no passado (até à década de 70 do século XX). A presente monitorização da estrutura populacional de A. atroparvus em Portugal, serve de base de caracterização dos últimos 15 anos. Recomenda-se a sua continuação no futuro, bem como a avaliação contínua de parâmetros ecológicos e biológicos em localidades-sentinela. Estas deverão incluir áreas com elevada pressão turística e/ou com populações humanas migrantes. Abstract With the present state of environmental and climate change, there is a growing concern about (re) emerging diseases, particularly those that are vector-borne. Of these, malaria is the vector-borne disease with the highest impact on human population. In a potential scenario of malaria resurgence in Europe, the rate of transmission of Plasmodium parasites will be most certainly affected by patterns of structure and connectivity among vector populations. This is the main subject of the present work, regarding the former malaria mosquito vector in Portugal, Anopheles atroparvus. Thus, we genotyped 10 microsatellite markers in mosquitoes collected at 10 sites in Portugal, between 1993 and 2008. Our data suggest a widespread gene flow over the country, and a positive correlation between geographical distances and genetic differentiation among A. atroparvus populations. Estimates of effective population size (Ne) based on temporal samples ranged between 1990 and 12197. Tests of "bottleneck effect" suggest a population contraction in almost all populations sampled. These results can be explained by the intensive anti-vector

Contribution of molecular markers to management options for Anaecypris hispanica, pp: 331-336. In: Conservation of freshwater fish: options for the future, Chapter 30, Blackwell Science, Oxford, UK, Jul 2002

Anaecypris hispanica (Steindachner) is restricted to the Guadiana River drainage, where its abund... more Anaecypris hispanica (Steindachner) is restricted to the Guadiana River drainage, where its abundance and geographical range have contracted markedly during the last 20 years. Recent ecological data suggest that the species is presently confined to ten tributaries in Portugal. Mitochondrial DNA (sequence and RFLP analyses of cytochrome b and control region) and nuclear DNA (five microsatellites loci) revealed strongly structured populations with substantial genetic variation. Using the mtDNA results, it was possible to define three ecologically significant units (ESUs): Odeleite, Foupana and a northern group comprising the Xévora, Caia, Álamo, Degebe, Ardila, Chança and Carreiras. Considering both microsatellite and mtDNA data, it is proposed that populations from the Xévora and Caia, Álamo, Degebe, Ardila, Chança and Carreiras plus Vascão, should be considered as six discrete management units (MUs) within the northern ESU. The more relevant conservation implications arising from the genetic results are: 1) translocations between the three ESUs should be avoided, at least in a short term; 2) active managed restocking actions are advised for populations under catastrophic reductions or extinctions, but the source of donor individuals have to be carefully evaluated; 3) in some rivers of the middle reaches, if the heterozygosity is very low, the use of admixed populations from the same or nearby MUs as broodstock can be an option.

Anais do Instituto de Higiene e Medicina Tropical, Chapter: I, Publisher: Instituto de Higiene e Medicina Tropical / Universidade Nova de Lisboa, pp.47-56

Currently, Tropical Medicine emerged as both an important medical specialty and scientific discip... more Currently, Tropical Medicine emerged as both an important medical specialty
and scientific discipline assuming a preponderant global dimension. Until a few years ago, several tropical pathologies, which were exclusively associated with tropical regions, have been gradually expanding to other geographic areas previously considered free of these infections. Vector-borne diseases are included among this group of infections. Actually, Europe
and the US struggle with disease outbreaks caused by pathogens primarily
associated with tropical regions. In fact, the impact of vector-borne diseases in human and veterinary health is increasingly being disseminated worldwide. The Institute of Hygiene and Tropical Medicine, University NOVA of Lisbon organized the 3rd National Congress of Tropical Medicine
and the 1st Congress of Portuguese Speaking Countries on Vector-borne Diseases, on 20th - 21st of April, 2015, dedicated to vector-borne diseases topics. The meeting was attended by about 300 participants from Portugal, Portuguese speaking countries, and other international scientific
centers. Overall, 57 oral communications and 41 posters were presented
on sessions that promoted great interest and scientific discussion, about the control, elimination, as well as the challenges associated with the development of novel methodologies applied to the diagnosis and treatment of several vector-borne diseases.

With the present state of environmental and climate change, there is a growing concern about (re)... more With the present state of environmental and climate change, there is a growing concern about (re) emerging diseases, particularly those that are vector-borne. Of these, malaria is the vector-borne disease with the highest impact on human population.
In a potential scenario of malaria resurgence in Europe, the rate of transmission of Plasmodium parasites will be most certainly affected by patterns of structure and connectivity among vector populations. This is the main subject of the present work, regarding the former malaria mosquito vector in Portugal, Anopheles atroparvus.
Thus, we genotyped 10 microsatellite markers in mosquitoes collected at 10 sites in Portugal, between 1993 and 2008.
Our data suggest a widespread gene flow over the country, and a positive correlation between geographical distances and genetic differentiation among A. atroparvus populations. Estimates of effective population size (Ne) based on temporal samples ranged between 1990 and 12197. Tests of "bottleneck effect" suggest a population contraction in almost all populations sampled. These results can be explained by the intensive anti-vector campaign taken in the past (until 1970s). This present population structure assessment of A. atroparvus in Portugal constitutes the background knowledge of the last 15 years.
We recommend that the assessment of ecological, biological and genetic parameters of sentinel-localities should be maintained in the future. These should include areas of tourism and with migrant human populations.

Salgueiro P. (2007) Genetic structure and gene flow of fragmented bat populations: consequences for conservation. PhD thesis, 161 p. Universidade de Lisboa. Lisboa, Dec 2007

http://repositorio.ul.pt/handle/10451/1685 ... more http://repositorio.ul.pt/handle/10451/1685

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Search in Bibioteca do Departamento de Biologia Animal da Faculdade de Ciências da Universidade d... more Search in Bibioteca do Departamento de Biologia Animal da Faculdade de Ciências da Universidade de Lisboa
https://ciencias.ulisboa.pt/pt/biologia-animal

Livro de Resumos do XVII Congresso Ibérico de Entomologia, p. 8.

Book of Abstracts of the 20th Conference of the European Society for Vector Ecology , p.70. DOI: 10.3920/978-90-8686-837-7

In recent years South America has been afflicted by successive high impact outbreaks of dengue, c... more In recent years South America has been afflicted by successive high impact outbreaks of dengue, chikungunya and zika. Aedes aegypti is the main mosquito vector of these diseases and the control of this mosquito is the sole solution to reduce transmission.
In order to improve vector control it is essential to study mosquito population dynamics. Here we present a population genetics study in a trans-border region in Amazonia between Brazil and French Guiana to provide further knowledge on these questions.
Two sites in French Guiana (SGO & CAY) were prospected for mosquito eggs in Dec. 2013, May-Jun. 2014 and Dec. 2014. Other two sites in the neighbour Amapá state in Brazil were also prospected in May-June 2014 (OIA & MAC). F0 adults from these collections were genotyped for 13 microsatellites to assess genetic diversity, population differentiation and effective population size. We also used a Bayesian clustering method to evaluate patterns of population structure.
Levels of genetic diversity and effective population size were comparable among the populations studied. Pairwise genetic differentiation over temporal samples was six fold lower than differentiation over distinct spatial samples collected in the same season. None of the pairwise comparisons between temporal samples was significantly differentiated. The most differentiated sample was MAC in Brazil with all comparisons being significant. A significant correlation was found between genetic and geographic distances (r=0.81, P=0.02), suggesting isolation by distance. We detected the presence of 3 clusters (k=3) congruent with CAY, SGO+OIA and MAC. The estimated number of migrants among samples was consistent with genetic differentiation results.
Results suggest: 1) High gene flow between the closest trans-border samples SGO in French Guiana and OIA in Brazil; 2) Barriers to gene flow between the detected clusters that may hinder the exchange of other genes of interest alleles (e.g. resistance alleles).

Livro de Resumos do 2º Congresso Nacional de Medicina Tropical

http://congressomedtrop.ihmt.unl.pt/files/Livro\_Resumos/index.html#/2/zoomed

INTRODUCTION Glossina palpalis gambiensis is the most common tsetse fly in Guinea-Bissau and a ma... more INTRODUCTION
Glossina palpalis gambiensis is the most common tsetse fly in Guinea-Bissau and a major vector of Human African Trypanosomiasis in West Africa (HAT) . Despite its widespread distribution, no vector-mediated HAT transmission has been reported in the country since the late 1970s. Wild populations of
the palpalis group display different levels of intraspecific variation that may influence vectorial capacity. Therefore, accurate knowledge on species identity and population structure is essential to predict the possible reestablishment and spread of HAT transmission in Guinea-Bissau.
METHODS
Genetic variation was analyzed in Glossina palpalis gambiensis samples from four districts of Guinea Bissau, using 11microsatellite loci. Three of the districts are in the mainland and one represents the insular part of the country.
RESULTS
Very low levels of genetic differentiation were observed among populations (FST = 0.06). This result is in agreement with a model-based clustering analysis that revealed the presence of a single population clustering grouping all samples regardless of geographic origin. These results suggest very little population substructure in G. palpalis gambiensis from this region.
CONCLUSIONS
Genetic evidence suggests considerable gene flow among G. palpalis gambiensis populations within mainland and between islands and mainland Guinea-Bissau. In the case of focal reestablishment of HAT transmission, the possibility of parasite dissemination through tsetse fly active dispersal should be taken
into account when planning vector control actions in Guinea Bissau.

Objectives: We have characterised the genetic diversity of Plasmodium falciparum populations in S... more Objectives: We have characterised the genetic diversity of Plasmodium falciparum populations in São Tomé and Príncipe islands (STP), West Africa, in order to assess patterns of population substructure and to relate these with epidemiological and control aspects of malaria.

Methods: Blood samples were collected from 3 localities of São Tomé and 1 from Príncipe in 2000 (rainy season). Plasmodium falciparum isolates identified by nested-PCR were genotyped for the surface antigen genes msp1 and msp2 and for genes associated with chloroquine-CQ (pfcrt) and pyrimethamine/sulfadoxine-SP (pfdhps, pfdhfr) resistance, by PCR-RFLP.

Results: 121 P. falciparum isolates were identified in 267 blood samples. Prevalence of infection was 44% in São Tomé and 49% in Príncipe. The 6-15 years-old group presented the highest prevalence.
Sixteen and 19 different alleles were obtained for msp-1 and msp-2, respectively. Genetic diversity was significantly higher in the northern sample of São Tomé. MOI values were comparable between islands (1.3-1.6) and highest in 6-15 year-olds.
The pfcrt K76T mutation related with CQ resistance was found in 97% of the isolates. All pfdhps and pfdhfr codons examined were polymorphic. The most frequent mutations at pfdhfr were: S108N (89%), N51I (85%) and C59R (60%). The triple mutation (51I/59R/108N) occurred in 45% of the samples. For pfdhps, the A437G mutation was found in 71% of isolates and the K540E in 3%. Four isolates (3%) carried the quintuple mutation (51I/59R/108N/437G/540E).

Conclusion: Both prevalence and MOI were higher in 6-15 year-olds, which may reflect lower immune status and higher infection exposure in this group. Genetic diversity at msp1 and msp2 was comparable with continental populations. Genetic differences within São Tomé suggest restrictions to gene flow coupled with different selective pressures between north and south of this island. The high frequency of mutations found in drug resistance genes agrees with high levels of resistance to CQ and SP observed in STP.

Abstract Book of Amazonian Conference on Emerging and Infectious Diseases (ACEID), p. 42.

Background: Aedes aegypti is vector of dengue and chikungunya viruses in French Guiana. A strong ... more Background: Aedes aegypti is vector of dengue and chikungunya viruses in French Guiana. A strong pyrethroid resistance has posed questions on the impact of vector control and resistance management. A prerequisite to improve those strategies is the investigation on population dynamics and factors which influence it. Therefore, we have implemented a population genetic study associated with deltamethrin resistance monitoring to provide further knowledge on these questions.
Material and Methods: Two sites of 1 km² in Saint-Georges de l’Oyapock (SGO) and Cayenne (CAY) were prospected for mosquito immature stages in December 2013. F0 females were genotyped for 13 microsatellites and for the knockdown resistance associated mutation V1016I. Genetic diversity and differentiation indices were obtained in Arlequin software. Genetic structure among samples was tested in the program STRUCTURE. Evolutionary scenario probabilities and estimates were computed in DIY-ABC.
Results: We detected the presence of two populations (k=2) congruent with each locality, with low but significant differentiation levels. Both populations exhibited similar effective population sizes and overall diversity. However, different evolutionary scenarios were obtained for each population. The V1016I kdr mutation is almost fixed in CAY population while SGO exhibited a larger proportion of heterozygotes.
Conclusion: Genetic differentiation between the two populations suggests barriers to gene flow that may limit the exchange of resistance alleles. Therefore, the expansion of insecticide resistance in each population may also be due to local selective pressure. Additional spatial and temporal investigations are being conducted to confirm this hypothesis.Background: Aedes aegypti is vector of dengue and chikungunya viruses in French Guiana. A strong pyrethroid resistance has posed questions on the impact of vector control and resistance management. A prerequisite to improve those strategies is the investigation on population dynamics and factors which influence it. Therefore, we have implemented a population genetic study associated with deltamethrin resistance monitoring to provide further knowledge on these questions.
Material and Methods: Two sites of 1 km² in Saint-Georges de l’Oyapock (SGO) and Cayenne (CAY) were prospected for mosquito immature stages in December 2013. F0 females were genotyped for 13 microsatellites and for the knockdown resistance associated mutation V1016I. Genetic diversity and differentiation indices were obtained in Arlequin software. Genetic structure among samples was tested in the program STRUCTURE. Evolutionary scenario probabilities and estimates were computed in DIY-ABC.
Results: We detected the presence of two populations (k=2) congruent with each locality, with low but significant differentiation levels. Both populations exhibited similar effective population sizes and overall diversity. However, different evolutionary scenarios were obtained for each population. The V1016I kdr mutation is almost fixed in CAY population while SGO exhibited a larger proportion of heterozygotes.
Conclusion: Genetic differentiation between the two populations suggests barriers to gene flow that may limit the exchange of resistance alleles. Therefore, the expansion of insecticide resistance in each population may also be due to local selective pressure. Additional spatial and temporal investigations are being conducted to confirm this hypothesis.

Abstract Book of the 19th International BioInformatics Workshop on Virus Evolution and Molecular Epidemiology, At National Institute for Infectious Diseases, p.91

Aedes aegypti is the most important mosquito vector of dengue and yellow fever, two of the most s... more Aedes aegypti is the most important mosquito vector of dengue and yellow fever, two of the most significant mosquito-borne viral diseases in the world. Due to the lack of antiviral therapy or vaccination, reducing dengue transmission depends only on mosquito control. In the archipelago of Cape Verde (West Africa) in November 2009 a dengue epidemic was declared with more than 20,000 people affected, 174 hemorrhagic fever cases and six deaths. The presence of Ae. aegypti in the archipelago was reported since 1931. In this study we aim to clarify the origins and population history of Ae. aegypti in Cape Verde by analyzing the variability of the mitochondrial gene NADH dehydrogenase subunit 4 (ND4). We analyzed 42 mosquitoes from three islands (Santiago, Brava and Fogo) collected before and after the dengue outbreak (2007/2010). ND4 sequences of 360bp revealed seven haplotypes, three of which are Cape Verde specific (haplotype diversity =0.637 and nucleotide diversity =0.002). All haplotypes were closely related and formed a star-like structure typical of expanded populations. In a comparison with 194 published haplotypes (including 6 African Aedes sp. outgroups), sequences from Cape Verde occurred in a basal clade mainly associated with West African mosquito populations. Our results suggest a high genetic diversity and a West African origin for this insular mosquito population. We found no evidence of new recent founder events that could be associated with the unique dengue outbreak in 2009.

Livro de Resumos das 5as Jornadas Científicas do IHMT

São Tomé e Príncipe (STP) é um arquipélago no Golfo da Guiné onde a tendência da prevalência de m... more São Tomé e Príncipe (STP) é um arquipélago no Golfo da Guiné onde a tendência da prevalência de malária tem sido muito irregular nas últimas décadas, com a tentativa de erradicação no início dos anos 80, para níveis meso-hiperendémicos durante a década de 90, de volta a um cenário de pré-eliminação na actualidade. É de extrema importância avaliar os efeitos de tais cenários epidemiológicos na estrutura genética e história demográfica do parasita P. falciparum. Assim, fomos analisar a diversidade genética de amostras de P. falciparum de STP obtidas desde 1997 até 2005.
Os principais objectivos deste trabalho foram: (1) avaliar os níveis de diferenciação genética entre populações de P. falciparum em STP, (2) efectuar estimativas do tamanho efectivo populacional destes parasitas; (3) comparar estes parâmetros durante o período de nove anos do estudo.
Cerca de 1.200 amostras de sangue foram colhidas em 1997, 2000, 2004 e 2005, em duas ilhas. Após a extracção do DNA, isolados de P. falciparum foram identificados por PCR. No total, foram genotipados 505 isolados de seis populações, através de 12 microssatélites neutros.
Encontrámos altos níveis de diversidade genética e níveis de diferenciação genética inter-populacional muito reduzida. Ao longo dos anos, detectou-se uma tendência de redução na percentagem de indivíduos infectados com P. falciparum. Esta tendência não se reflectiu no tamanho efectivo populacional estimado do parasita (Ne), cujas estimativas são comparáveis ou mesmo superior às obtidas em amostras continentais. Não se detectou diferenciação genética significativa entre as ilhas.
Estes resultados confirmam o intenso nível de transmissão da malária em São Tomé e Príncipe nas últimas décadas. Apesar da condição insular de STP, estes níveis de transmissão têm sido tão elevados e estáveis ao longo dos anos, que não há um sinal genético actual do isolamento geográfico em comparação com regiões homólogas no continente.

DOI: 10.13140/2.1.4768.0327

Introdução: Nas ilhas de São Tomé e Príncipe (STP), a malária atinge níveis meso-hiperendémicos,... more Introdução:
Nas ilhas de São Tomé e Príncipe (STP), a malária atinge níveis meso-hiperendémicos, com predominância de Plasmodium falciparum. Em 1980-1982, decorreu um programa de erradicação no qual foi utilizado o fármaco cloroquina. Mais recentemente (2000), iniciou-se um novo esquema terapêutico à base de derivados da artemisinina. A informação acerca da estrutura genética de populações geograficamente isoladas de P. falciparum pode ter importantes repercussões no controlo da malária.

Objectivos:
Caracterizou-se a variabilidade genética de populações de P. falciparum em STP, utilizando microssatélites, de forma a: 1) comparar os níveis de variabilidade e diferenciação genética entre amostras colhidas em dois anos diferentes (2000 e 2004); 2) avaliar o possível impacto da introdução de um novo fármaco na estrutura populacional parasitária.

Material e Métodos:
Foram analisadas 355 amostras sanguíneas de habitantes de duas localidades (Neves, São Tomé; Rua dos Trabalhadores, Príncipe), colhidas nos anos de 2000 e 2004. Após extracção de DNA, foram identificados isolados de P. falciparum por PCR. Estes foram analisados para 12 loci de microssatélites. A análise das frequências genéticas foi efectuada com o programa GENEPOP e FSTAT.

Resultados:
No total foram identificados 114 isolados de P. falciparum. Houve uma acentuada diminuição da prevalência de infecção 49% a 10% no Príncipe. A diversidade genética foi comparável entre ilhas. Na comparação entre anos, verificou-se uma diminuição da diversidade genética de 2000 para 2004, no Príncipe (2000: Rs=5/He=0.82; 2004: Rs=4; He=0.76). Foram detectados níveis de diferenciação genética significativos, estimados pelo parâmetro Fst, entre ilhas e entre anos diferentes (mesma ilha).

Conclusões:
Os níveis de diferenciação genética observados entre ilhas sugerem a ocorrência de restrições ao fluxo genético, possivelmente associadas à barreira oceânica e consequente isolamento geográfico destas populações parasitárias insulares. A redução de diversidade genética entre 2000 e 2004 observada na ilha do Príncipe poderá reflectir uma redução populacional associada à implementação de medidas de controlo da malária.

Este trabalho foi financiado pelo Programa POCI2010, Fundação para a Ciência e a Tecnologia, Portugal (POCI/SAU-ESP/56903/2004)

http://www.eurnegvec.org/2ac\_abstractbook.pdf

The major malaria vector Anopheles gambiae s.s. displays strongpopulation subdivision across su... more The major malaria vector Anopheles gambiae s.s. displays strongpopulation subdivision across sub-Saharan Africa. In West Africa, twomolecular forms have been described and are considered units of incipientspeciation, albeit with varying levels of inter-form gene ﬂow alongtheir sympatric distribution range. However, studies on molecular formdifferentiation often analyzed samples from relatively few localities orregions. To provide an overall picture of the population structure of An. gambiae s.s. in west Africa, we have genotyped 25 samples, obtainedmostly by indoor resting collections, from 12 African countries for 13 microsatellites on chromosome-3. Our area-wide results conﬁrm a cleargenetic differentiation between M and S forms using loci outside genomicregions of highest divergence. Furthermore, both Bayesian clustering andprincipal components analyses revealed further population substructuringin the M-form, with samples from Western Africa (from The Gambia toNigeria) forming a distinct genetic cluster from those of West-CentralAfrica (from Cameroon to Angola). This subdivision is likely to beassociated with the Forest-savannah ecosystem transition coupled withthe accumulation of polymorphic chromosomal inversions in this vector species.

Abstract Book of Amazonian Conference on Emerging and Infectious Diseases (ACEID), p. 41.

DOI: 10.13140/2.1.4243.7449

São Tome and Principe (STP) is a small nation – archipelago in West Africa where the trend of mal... more São Tome and Principe (STP) is a small nation – archipelago in West Africa where the trend of malaria prevalence has been changing back and forth, in the last decades: from an unattempted near eradication in the early 80s, to meso-hyperendemic levels in the 90s, back to pre-elimination in present times. It is of utmost importance to evaluate the effects of such epidemiological scenarios on the population genetic structure and demographic history of the parasite P. falciparum. Therefore, we have analyzed the genetic diversity of P. falciparum samples from STP collected since 1997 until 2005.

Our main goals are: (1) to examine the levels of genetic differentiation among populations; (2) to estimate the effective population size; (3) to compare those parameters over a nine years period.

Around 1200 blood samples were obtained during active case detection surveys in 1997 2000, 2004 and 2005, in two islands. After DNA extraction, P. falciparum isolates were identified by PCR. Overall, we genotyped 505 isolates from six populations, using 12 neutral microsatellite loci.

We have found high levels of genetic diversity and minimal inter-population genetic differentiation. Over the years, we detected a declining trend in the percentage of individuals infected with P. falciparum that did not correlate with the parasite effective population size (Ne). Ne estimates were similar or higher than in other mainland African samples. No significant genetic differentiation was found between islands.
In spite of the insular condition of these populations, levels of parasite transmission have been so high and stable over the times that there is no current genetic signature of geographic isolation, comparing with homologous continental regions.

https://www.researchgate.net/publication/321385976\_REPORT\_on\_the\_Workshop\_on\_Malaria\_Transmission\_current\_challenges\_and\_new\_tools\_in\_the\_elimination\_context, 2017

The Workshop on Malaria Transmission: current challenges and new tools in the elimination context... more The Workshop on Malaria Transmission: current challenges and new tools in the elimination context was held at the Instituto de Higiene e Medicina Tropical (IHMT) (http://www.ihmt.unl.pt/workshop-malaria-transmission/) between the 30th and 31st of October 2017, under the scientific project of the Centre for Global Health and Tropical Medicine (GHTM). IHMT/ GHTM aimed at creating an in depth debate over new challenges and innovative tools in the current elimination context of malaria transmission in the world. Specifically, the conference explored the environmental and social determinants in the new malaria transmission contexts, as well as current approaches and new tools to measure malaria transmission or efficacy of control methods. We targeted Health professionals, Social, Biomedical and Geographical Science researchers or students and any one with interest in Malaria. Having this in mind, our Workshop included five plenary sessions with distinguished speakers from various international institutions. The topics were as follows: • Geospatial modelling of changes to vector populations following insecticide-based malaria control • Current approaches and new tools to measure malaria transmission • The economic challenges and benefits of eliminating malaria • Impact of genetic and bio-ecological heterogeneities of mosquito vectors on Malaria transmission and control in Africa: future perspectives in context of elimination and growing urbanization • Beyond 2020: Pushing the transmission-blocking envelope of malaria elimination tools

http://www.icnf.pt/portal/naturaclas/patrinatur/resource/gen/pipistrellus-pt