Detection of Plasmodium falciparum male and female gametocytes and determination of parasite sex ratio in human endemic populations by novel, cheap and robust RTqPCR assays

The presence of Plasmodium falciparum gametocytes in peripheral blood is essential for human to mosquito parasite transmission. The detection of submicroscopic infections with gametocytes and the estimation of the gametocyte sex ratio are crucial to assess the human host potential ability to infect mosquitoes and transmit malaria parasites

Insertion polymorphisms of SINE200 retrotransposons within speciation islands of Anopheles gambiae molecular forms

<p>Abstract</p> <p>Background</p> <p>SINEs (Short INterspersed Elements) are homoplasy-free and co-dominant genetic markers which are considered to represent useful tools for population genetic studies, and could help clarifying the speciation processes ongoing within the major malaria vector in Africa, <it>Anopheles gambiae </it>s.s. Here, we report the results of the analysis of the insertion polymorphism of a nearly 200 bp-long SINE (<it>SINE200</it>) within genome areas of high differentiation (i.e. "speciation islands") of M and S <it>A. gambiae </it>molecular forms.</p> <p>Methods</p> <p>A <it>SINE</it>-PCR approach was carried out on thirteen <it>SINE200 </it>insertions in M and S females collected along the whole range of distribution of <it>A. gambiae </it>s.s. in sub-Saharan Africa. Ten specimens each for <it>Anopheles arabiensis</it>, <it>Anopheles melas, Anopheles quadriannulatus </it>A and 15 M/S hybrids from laboratory crosses were also analysed.</p> <p>Results</p> <p>Eight loci were successfully amplified and were found to be specific for <it>A. gambiae </it>s.s.: 5 on 2L chromosome and one on X chromosome resulted monomorphic, while two loci positioned respectively on 2R (i.e. <it>S200 </it>2R12D) and X (i.e. <it>S200 </it>X6.1) chromosomes were found to be polymorphic. <it>S200 </it>2R12D was homozygote for the insertion in most S-form samples, while intermediate levels of polymorphism were shown in M-form, resulting in an overall high degree of genetic differentiation between molecular forms (Fst = 0.46 p < 0.001) and within M-form (Fst = 0.46 p < 0.001). The insertion of <it>S200 </it>X6.1 was found to be fixed in all M- and absent in all S-specimens. This led to develop a novel easy-to-use PCR approach to straightforwardly identify <it>A. gambiae </it>molecular forms. This novel approach allows to overcome the constraints associated with markers on the rDNA region commonly used for M and S identification. In fact, it is based on a single copy and irreversible <it>SINE200 </it>insertion and, thus, is not subjected to peculiar evolutionary patterns affecting rDNA markers, e.g. incomplete homogenization of the arrays through concerted evolution and/or mixtures of M and S IGS-sequences among the arrays of single chromatids.</p> <p>Conclusion</p> <p>The approach utilized allowed to develop new easy-to-use co-dominant markers for the analysis of genetic differentiation between M and S-forms and opens new perspectives in the study of the speciation process ongoing within <it>A. gambiae</it>.</p

Novel cDNAs encoding salivary proteins from the malaria vector Anopheles gambiae

AbstractSeveral genes encoding salivary components of the mosquito Anopheles gambiae were identified using a selective trapping approach. Among these, five corresponded to genes expressed specifically in female glands and their role may possibly be linked to blood-feeding. Our collection included a fourth member of the D7 protein family and two polypeptides that showed weak similarity to anti-coagulants from distantly related species. Moreover, we identified two additional members of a novel group of proteins that we named glandins. The isolation of tissue-specific genes represents a first step toward a deeper molecular analysis of mosquito salivary secretions

Comparative analyses reveal discrepancies among results of commonly used methods for Anopheles gambiaemolecular form identification

<p>Abstract</p> <p>Background</p> <p><it>Anopheles gambiae </it>M and S molecular forms, the major malaria vectors in the Afro-tropical region, are ongoing a process of ecological diversification and adaptive lineage splitting, which is affecting malaria transmission and vector control strategies in West Africa. These two incipient species are defined on the basis of single nucleotide differences in the IGS and ITS regions of multicopy rDNA located on the X-chromosome. A number of PCR and PCR-RFLP approaches based on form-specific SNPs in the IGS region are used for M and S identification. Moreover, a PCR-method to detect the M-specific insertion of a short interspersed transposable element (<it>SINE200</it>) has recently been introduced as an alternative identification approach. However, a large-scale comparative analysis of four widely used PCR or PCR-RFLP genotyping methods for M and S identification was never carried out to evaluate whether they could be used interchangeably, as commonly assumed.</p> <p>Results</p> <p>The genotyping of more than 400 <it>A. gambiae </it>specimens from nine African countries, and the sequencing of the IGS-amplicon of 115 of them, highlighted discrepancies among results obtained by the different approaches due to different kinds of biases, which may result in an overestimation of MS putative hybrids, as follows: i) incorrect match of M and S specific primers used in the allele specific-PCR approach; ii) presence of polymorphisms in the recognition sequence of restriction enzymes used in the PCR-RFLP approaches; iii) incomplete cleavage during the restriction reactions; iv) presence of different copy numbers of M and S-specific IGS-arrays in single individuals in areas of secondary contact between the two forms.</p> <p>Conclusions</p> <p>The results reveal that the PCR and PCR-RFLP approaches most commonly utilized to identify <it>A. gambiae </it>M and S forms are not fully interchangeable as usually assumed, and highlight limits of the actual definition of the two molecular forms, which might not fully correspond to the two <it>A. gambiae </it>incipient species in their entire geographical range. These limits are discussed and operational suggestions on the choice of the most convenient method for large-scale M- and S-form identification are provided, also taking into consideration technical aspects related to the epidemiological characteristics of different study areas.</p

Exploring the origin and degree of genetic isolation of Anopheles gambiae from the islands of São Tomé and Príncipe, potential sites for testing transgenic-based vector control

The evolutionary processes at play between island and mainland populations of the malaria mosquito vector Anopheles gambiae sensu stricto are of great interest as islands may be suitable sites for preliminary application of transgenic-based vector control strategies. São Tomé and Príncipe, located off the West African coast, have received such attention in recent years. This study investigates the degree of isolation of An. gambiae s.s. populations between these islands and the mainland based on mitochondrial and ribosomal DNA molecular data. We identify possible continental localities from which these island populations derived. For these purposes, we used FST values, haplotype networks, and nested clade analysis to estimate migration rates and patterns. Haplotypes from both markers are geographically widespread across the African continent. Results indicate that the populations from São Tomé and Príncipe are relatively isolated from continental African populations, suggesting they are promising sites for test releases of transgenic individuals. These island populations are possibly derived from two separate continental migrations. This result is discussed in the context of the history of the African slave trade with respect to São Tomé and Príncipe

Remarkable diversity of intron-1 of the para voltage-gated sodium channel gene in an Anopheles gambiae/Anopheles coluzzii hybrid zone.

BACKGROUND: Genomic differentiation between Anopheles gambiae and Anopheles coluzzii--the major malaria vectors in sub-Saharan Africa--is localized into large "islands" toward the centromeres of chromosome-X and the two autosomes. Linkage disequilibrium between these genomic islands was first detected between species-specific polymorphisms within ribosomal DNA genes (IGS-rDNA) on the X-chromosome and a single variant at position 702 of intron 1 (Int-1702) of the para Voltage-Gated Sodium Channel (VGSC) gene on chromosome arm 2 L. Intron-1 sequence data from West and Central Africa revealed two clearly distinct and species-specific haplogroups, each characterized by very low polymorphism, which has been attributed to a selective sweep. The aim of this study was to analyse Int-1 sequence diversity in A. gambiae and A. coluzzii populations from the Far-West of their range, in order to assess whether this selective-sweep signature could persist in a zone of high interspecific hybridization. METHODS: A 531 bp region of VGSC Int-1 was sequenced in 21 A. coluzzii, 31 A. gambiae, and 12 hybrids from The Gambia and Guinea Bissau, located within the Far-West geographical region, and in 53 A. gambiae s.l. samples from the rest of the range. RESULTS: Far-West samples exhibit dramatic Int-1 polymorphism, far higher within each country than observed throughout the rest of the species range. Moreover, patterning of haplotypes within A. coluzzii confirms previous evidence of a macro-geographic subdivision into a West and a Central African genetic cluster, and reveals a possible genetic distinction of A. coluzzii populations from the Far-West. CONCLUSIONS: The results suggest a relaxation of selective pressures acting across the VGSC gene region in the hybrid zone. Genetic differentiation in the Far-West could be attributable to a founder effect within A. coluzzii, with subsequent extensive gene flow with secondarily-colonizing A. gambiae, potentially yielding a novel insight on the dynamic processes impacting genetic divergence of these key malaria vectors

Distribution of knock-down resistance mutations in Anopheles gambiae molecular forms in west and west-central Africa

<p>Abstract</p> <p>Background</p> <p><it>Knock-down </it>resistance (<it>kdr</it>) to DDT and pyrethroids in the major Afrotropical vector species, <it>Anopheles gambiae </it>sensu stricto, is associated with two alternative point mutations at amino acid position 1014 of the voltage-gated sodium channel gene, resulting in either a leucine-phenylalanine (L1014F), or a leucine-serine (L1014S) substitution. In <it>An. gambiae </it>S-form populations, the former mutation appears to be widespread in west Africa and has been recently reported from Uganda, while the latter, originally recorded in Kenya, has been recently found in Gabon, Cameroon and Equatorial Guinea. In M-form populations surveyed to date, only the L1014F mutation has been found, although less widespread and at lower frequencies than in sympatric S-form populations.</p> <p>Methods</p> <p><it>Anopheles gambiae </it>M- and S-form specimens from 19 sites from 11 west and west-central African countries were identified to molecular form and genotyped at the <it>kdr </it>locus either by Hot Oligonucleotide Ligation Assay (HOLA) or allele-specific PCR (AS-PCR).</p> <p>Results</p> <p>The <it>kdr </it>genotype was determined for about 1,000 <it>An. gambiae </it>specimens. The L1014F allele was found at frequencies ranging from 6% to 100% in all S-form samples (N = 628), with the exception of two samples from Angola, where it was absent, and coexisted with the L1014S allele in samples from Cameroon, Gabon and north-western Angola. The L1014F allele was present in M-form samples (N = 354) from Benin, Nigeria, and Cameroon, where both M- and S-forms were sympatric.</p> <p>Conclusion</p> <p>The results represent the most comprehensive effort to analyse the overall distribution of the L1014F and L1014S mutations in <it>An. gambiae </it>molecular forms, and will serve as baseline data for resistance monitoring. The overall picture shows that the emergence and spread of <it>kdr </it>alleles in <it>An. gambiae </it>is a dynamic process and that there is marked intra- and inter-form heterogeneity in resistance allele frequencies. Further studies are needed to determine: i) the importance of selection pressure exerted by both agricultural and public health use of pyrethroid insecticides, ii) the phenotypic effects, particularly when the two mutations co-occur; and iii) the epidemiological importance of <it>kdr </it>for both pyrethroid- and DDT-based malaria control operations, particularly if/when the two insecticides are to be used in concert.</p

Novel Insights Into the Protective Role of Hemoglobin S and C Against Plasmodium falciparum Parasitemia.

Although hemoglobin S (HbS) and hemoglobin C (HbC) are well known to protect against severe Plasmodium falciparum malaria, conclusive evidence on their role against infection has not yet been obtained. Here we show, in 2 populations from Burkina Faso (2007-2008), that HbS is associated with a 70% reduction of harboring P. falciparum parasitemia at the heterozygous state (odds ratio [OR] for AS vs AA, 0.27; 95% confidence interval [CI], .11-.66; P = .004). There is no evidence of protection for HbC in the heterozygous state (OR for AC vs AA, 1.49; 95% CI, .69-3.21; P = .31), whereas protection even higher than that observed with AS is observed in the homozygous and double heterozygous states (OR for CC + SC vs AA, 0.04; 95% CI, .01-.29; P = .002). The abnormal display of parasite-adhesive molecules on the surface of HbS and HbC infected erythrocytes, disrupting the pathogenic process of sequestration, might displace the parasite from the deep to the peripheral circulation, promoting its elimination at the spleen level

The “Far-West” of Anopheles gambiae Molecular Forms

The main Afrotropical malaria vector, Anopheles gambiae sensu stricto, is undergoing a process of sympatric ecological diversification leading to at least two incipient species (the M and S molecular forms) showing heterogeneous levels of divergence across the genome. The physically unlinked centromeric regions on all three chromosomes of these closely related taxa contain fixed nucleotide differences which have been found in nearly complete linkage disequilibrium in geographic areas of no or low M-S hybridization. Assays diagnostic for SNP and structural differences between M and S forms in the three centromeric regions were applied in samples from the western extreme of their range of sympatry, the only area where high frequencies of putative M/S hybrids have been reported. The results reveal a level of admixture not observed in the rest of the range. In particular, we found: i) heterozygous genotypes at each marker, although at frequencies lower than expected under panmixia; ii) virtually all possible genotypic combinations between markers on different chromosomes, although genetic association was nevertheless detected; iii) discordant M and S genotypes at two X-linked markers near the centromere, suggestive of introgression and inter-locus recombination. These results could be indicative either of a secondary contact zone between M and S, or of the maintenance of ancestral polymorphisms. This issue and the perspectives opened by these results in the study of the M and S incipient speciation process are discussed

Cystic echinococcosis in northern Tanzania: a pilot study in Maasai livestock-keeping communities

Background: There are close similarities between the life-cycles of Echinococcus granulosus sensu lato (E. granulosus s.l.) that causes cystic echinococcosis (CE) in humans and Taenia multiceps/Coenurus cerebralis that causes cerebral coenurosis in small ruminants. Recent evidence highlights that livestock in Maasai communities of northern Tanzania are suffering from increases in the prevalence of cerebral coenurosis, leading to concerns about a possible concurrent increased risk of human CE. The aim of this study was to estimate the prevalence of human abdominal CE and the prevalence and species/genotypes of E. granulosus s.l. in livestock in Maasai communities. Methods: Human CE was diagnosed by abdominal ultrasound on volunteers aged ≥ 7 years in five villages in the Longido and Ngorongoro Districts in northern Tanzania. Infection in ruminants was evaluated through inspection in local abattoirs, followed by molecular identification of one cyst per animal, with a priority for hepatic cysts, using PCR targeting of the cytochrome c oxidase I gene (COX1), followed by restriction fragment length polymorphism and multiplex PCR, and sequencing of non-E. granulosus s.l. samples. Results: Ultrasound was performed on 823 volunteers (n = 352 in two villages in Longido District, and n = 471 in three villages of Ngorongoro). Hepatic CE cases were diagnosed only in Ngorongoro (n = 6; 1.3%), of which three had active cysts. Village-level prevalence of CE ranged between 0 and 2.4%. Of the 697 ruminants inspected, 34.4% had parasitic cysts. Molecular identification was achieved for 140 of the 219 (63.9%) cysts sampled. E. granulosus s.l. and T. hydatigena/Cysticercus tenuicollis were identified in 51.4% and 48.6%, respectively, of livestock cysts. E. granulosus s.l. was identified in livestock from both Longido (35.3% of 116 genotyped cysts) and Ngorongoro (91.2% of 34 genotyped cysts). Of the total of 72 E. granuslosus s.l. cysts identified in livestock, 87.5% were E. granulosus sensu stricto (G1–G3 genotypes), 9.7% were E. ortleppi (G5) and one cyst was E. canadensis (G6–10). The three active human cysts, which were removed surgically, were G1–G3 genotypes. Conclusions: Multiple species/genotypes of E. granulosus s.l. are circulating in Maasai communities of northern Tanzania. Human CE was detected in villages of Ngorongoro District and a high prevalence of echinococcal cysts was observed in livestock in both districts. More precise estimation of the prevalence in this area and a better understanding of the specific risk factors for CE among Maasai communities in northern Tanzania is needed. Interventions targeting transmission routes common to both E. granulosus s.l. and T. multiceps would have dual benefits for preventing both human and livestock disease