Microsatellite data suggest significant population structure and differentiation within the malaria vector Anopheles darlingi in Central and South America

Background: Anopheles darlingi is the most important malaria vector in the Neotropics. An understanding of A. darlingi's population structure and contemporary gene flow patterns is necessary if vector populations are to be successfully controlled. We assessed population genetic structure and levels of differentiation based on 1,376 samples from 31 localities throughout the Peruvian and Brazilian Amazon and Central America using 5-8 microsatellite loci.Results: We found high levels of polymorphism for all of the Amazonian populations (mean R-S = 7.62, mean H-O = 0.742), and low levels for the Belize and Guatemalan populations (mean R-S = 4.3, mean H-O = 0.457). The Bayesian clustering analysis revealed five population clusters: northeastern Amazonian Brazil, southeastern and central Amazonian Brazil, western and central Amazonian Brazil, Peruvian Amazon, and the Central American populations. Within Central America there was low nonsignificant differentiation, except for between the populations separated by the Maya Mountains. Within Amazonia there was a moderate level of significant differentiation attributed to isolation by distance. Within Peru there was no significant population structure and low differentiation, and some evidence of a population expansion. The pairwise estimates of genetic differentiation between Central America and Amazonian populations were all very high and highly significant (F-ST = 0.1859-0.3901, P < 0.05). Both the D-A and F-ST distance-based trees illustrated the main division to be between Central America and Amazonia.Conclusion: We detected a large amount of population structure in Amazonia, with three population clusters within Brazil and one including the Peru populations. The considerable differences in N-e among the populations may have contributed to the observed genetic differentiation. All of the data suggest that the primary division within A. darlingi corresponds to two white gene genotypes between Amazonia (genotype 1) and Central America, parts of Colombia and Venezuela (genotype 2), and are in agreement with previously published mitochondrial COI gene sequences interpreted as incipient species. Overall, it appears that two main factors have contributed to the genetic differentiation between the population clusters: physical distance between the populations and the differences in effective population sizes among the subpopulations

Plasmodium vivax circumsporozoite genotypes: a limited variation or new subspecies with major biological consequences?

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium vivax </it>circumsporozoite variants have been identified in several geographical areas. The real implication of the genetic variation in this region of the <it>P. vivax </it>genome has been questioned for a long time. Although previous studies have observed significant association between VK210 and the Duffy blood group, we present here that evidences of this variation are limited to the CSP central portion.</p> <p>Methods</p> <p>The phylogenetic analyses were accomplished starting from the amplification of conserved domains of <it>18 SSU RNAr </it>and <it>Cyt B</it>. The antibodies responses against the CSP peptides, MSP-1, AMA-1 and DBP were detected by ELISA, in plasma samples of individuals infected with two <it>P. vivax CS </it>genotypes: VK210 and <it>P. vivax</it>-like.</p> <p>Results</p> <p>These analyses of the two markers demonstrate high similarity among the <it>P. vivax CS </it>genotypes and surprisingly showed diversity equal to zero between VK210 and <it>P. vivax</it>-like, positioning these <it>CS </it>genotypes in the same clade. A high frequency IgG antibody against the N- and C-terminal regions of the <it>P. vivax </it>CSP was found as compared to the immune response to the R- and V- repetitive regions (<it>p </it>= 0.0005, Fisher's Exact test). This difference was more pronounced when the <it>P. vivax</it>-like variant was present in the infection (<it>p </it>= 0.003, Fisher's Exact test). A high frequency of antibody response against MSP-1 and AMA-1 peptides was observed for all <it>P. vivax CS </it>genotypes in comparison to the same frequency for DBP.</p> <p>Conclusions</p> <p>This results target that the differences among the <it>P. vivax CS </it>variants are restrict to the central repeated region of the protein, mostly nucleotide variation with important serological consequences.</p

Increased interleukin-10 and interferon-γ levels in Plasmodium vivax malaria suggest a reciprocal regulation which is not altered by IL-10 gene promoter polymorphism

Background\ud In human malaria, the naturally-acquired immune response can result in either the elimination of the parasite or a persistent response mediated by cytokines that leads to immunopathology. The cytokines are responsible for all the symptoms, pathological alterations and the outcome of the infection depends on the reciprocal regulation of the pro and anti-inflammatory cytokines. IL-10 and IFN-gamma are able to mediate this process and their production can be affected by single nucleotide polymorphisms (SNPs) on gene of these cytokines. In this study, the relationship between cytokine IL-10/IFN-gamma levels, parasitaemia, and their gene polymorphisms was examined and the participation of pro-inflammatory and regulatory balance during a natural immune response in Plasmodium vivax-infected individuals was observed.\ud \ud Methods\ud The serum levels of the cytokines IL-4, IL-12, IFN-gamma and IL-10 from 132 patients were evaluated by indirect enzyme-linked immunosorbent assays (ELISA). The polymorphism at position +874 of the IFN-gamma gene was identified by allele-specific polymerase chain reaction (ASO-PCR) method, and the polymorphism at position -1082 of the IL-10 gene was analysed by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism).\ud \ud Results\ud The levels of a pro- (IFN-gamma) and an anti-inflammatory cytokine (IL-10) were significantly higher in P. vivax-infected individuals as compared to healthy controls. The IFN-gamma levels in primoinfected patients were significantly higher than in patients who had suffered only one and more than one previous episode. The mutant alleles of both IFN-gamma and IL-10 genes were more frequent than the wild allele. In the case of the IFNG+874 polymorphism (IFN-gamma) the frequencies of the mutant (A) and wild (T) alleles were 70.13% and 29.87%, respectively. Similar frequencies were recorded in IL-10-1082, with the mutant (A) allele returning a frequency of 70.78%, and the wild (G) allele a frequency of 29.22%. The frequencies of the alleles associated with reduced production of both IFN-gamma and IL-10 were high, but this effect was only observed in the production of IFN-gamma.\ud \ud Conclusions\ud This study has shown evidence of reciprocal regulation of the levels of IL-10 and IFN-gamma cytokines in P. vivax malaria, which is not altered by the presence of polymorphism in the IL-10 gene.This research was funded by the National Research Council from Brazil (CNPq-620139/2006-4) and Fundação de Amparo à pesquisa do Estado do Pará (FAPESPA- 042/2007) with financial support to MGC, scholarships from CNPq to SPTC and FAPESPA to TSM. The authors thank Fundação Centro de Hemoterapia e Hematologia do Pará (HEMOPA) for providing the control group samples. We thank Julius C. Haffala for reviewing the manuscript

Correction: Serologically Defined Variations in Malaria Endemicity in Pará State, Brazil.

[This corrects the article DOI: 10.1371/journal.pone.0113357.]

Phylogeography of the neotropical Anopheles triannulatus complex (Diptera: Culicidae) supports deep structure and complex patterns

Abstract Background The molecular phylogenetic relationships and population structure of the species of the Anopheles triannulatus complex: Anopheles triannulatus s.s., Anopheles halophylus and the putative species Anopheles triannulatus C were investigated. Methods\ud \ud The mitochondrial COI gene, the nuclear white gene and rDNA ITS2 of samples that include the known geographic distribution of these taxa were analyzed. Phylogenetic analyses were performed using Bayesian inference, Maximum parsimony and Maximum likelihood approaches. Results Each data set analyzed septely yielded a different topology but none provided evidence for the seption of An. halophylus and An. triannulatus C, consistent with the hypothesis that the two are undergoing incipient speciation. The phylogenetic analyses of the white gene found three main clades, whereas the statistical parsimony network detected only a single metapopulation of Anopheles triannulatus s.l. Seven COI lineages were detected by phylogenetic and network analysis. In contrast, the network, but not the phylogenetic analyses, strongly supported three ITS2 groups. Combined data analyses provided the best resolution of the trees, with two major clades, Amazonian (clade I) and trans-Andean + Amazon Delta (clade II). Clade I consists of multiple subclades: An. halophylus + An. triannulatus C; trans-Andean Venezuela; central Amazonia + central Bolivia; Atlantic coastal lowland; and Amazon delta. Clade II includes three subclades: Panama; cis-Andean Colombia; and cis-Venezuela. The Amazon delta specimens are in both clades, likely indicating local sympatry. Spatial and molecular variance analyses detected nine groups, corroborating some of subclades obtained in the combined data analysis. Conclusion Combination of the three molecular markers provided the best resolution for differentiation within An. triannulatus s.s. and An. halophylus and C. The latest two species seem to be very closely related and the analyses performed were not conclusive regarding species differentiation. Further studies including new molecular markers would be desirable to solve this species status question. Besides, results of the study indicate a trans-Andean origin for An. triannulatus s.l. The potential implications for malaria epidemiology remain to be investigated.This project has been financially supported by NIH grant AI ROI 54139–02 to JE Conn, PAPES IV Fiocruz-CNPq to RLO, and from the Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (Grant 2011/20397-7 to MAMS), CNPq (BPP no. 301666/2011-3 to MAMS).This research was performed under a Memorandum of Understanding between the Walter Reed Army Institute of Research and the Smithsonian Institution, with institutional support provided by both organizations. The material to be published reflects the views of the authors and should not be construed to represent those of the Department of the Army or the Department of Defense

Phylogeography of the neotropical Anopheles triannulatus complex (Diptera: Culicidae) supports deep structure and complex patterns

Abstract Background The molecular phylogenetic relationships and population structure of the species of the Anopheles triannulatus complex: Anopheles triannulatus s.s., Anopheles halophylus and the putative species Anopheles triannulatus C were investigated. Methods\ud \ud The mitochondrial COI gene, the nuclear white gene and rDNA ITS2 of samples that include the known geographic distribution of these taxa were analyzed. Phylogenetic analyses were performed using Bayesian inference, Maximum parsimony and Maximum likelihood approaches. Results Each data set analyzed septely yielded a different topology but none provided evidence for the seption of An. halophylus and An. triannulatus C, consistent with the hypothesis that the two are undergoing incipient speciation. The phylogenetic analyses of the white gene found three main clades, whereas the statistical parsimony network detected only a single metapopulation of Anopheles triannulatus s.l. Seven COI lineages were detected by phylogenetic and network analysis. In contrast, the network, but not the phylogenetic analyses, strongly supported three ITS2 groups. Combined data analyses provided the best resolution of the trees, with two major clades, Amazonian (clade I) and trans-Andean + Amazon Delta (clade II). Clade I consists of multiple subclades: An. halophylus + An. triannulatus C; trans-Andean Venezuela; central Amazonia + central Bolivia; Atlantic coastal lowland; and Amazon delta. Clade II includes three subclades: Panama; cis-Andean Colombia; and cis-Venezuela. The Amazon delta specimens are in both clades, likely indicating local sympatry. Spatial and molecular variance analyses detected nine groups, corroborating some of subclades obtained in the combined data analysis. Conclusion Combination of the three molecular markers provided the best resolution for differentiation within An. triannulatus s.s. and An. halophylus and C. The latest two species seem to be very closely related and the analyses performed were not conclusive regarding species differentiation. Further studies including new molecular markers would be desirable to solve this species status question. Besides, results of the study indicate a trans-Andean origin for An. triannulatus s.l. The potential implications for malaria epidemiology remain to be investigated.This project has been financially supported by NIH grant AI ROI 54139–02 to JE Conn, PAPES IV Fiocruz-CNPq to RLO, and from the Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (Grant 2011/20397-7 to MAMS), CNPq (BPP no. 301666/2011-3 to MAMS).This research was performed under a Memorandum of Understanding between the Walter Reed Army Institute of Research and the Smithsonian Institution, with institutional support provided by both organizations. The material to be published reflects the views of the authors and should not be construed to represent those of the Department of the Army or the Department of Defense