Phylogeography of the neotropical sand fly Lutzomyia longipalpis inferred from mitochondrial DNA sequences.

Sand flies in the Lutzomyia longipalpis species complex include the primary vector of Leishmania chagasi, the etiologic agent of visceral leishmaniasis in the Neotropics. Twelve L. longipalpis populations from South and Central America were compared using the cytochrome c oxidase I (COI) gene from the mitochondrial genome. The haplotype profiles for each population revealed that the majority of sequence variation was inter-population (98%) rather than intra-population, suggesting that sequence polymorphisms at the COI locus should provide excellent characters for the study of phylogenetic relationships among populations. Phylogenetic reconstruction using distance (neighbor-joining) and maximum parsimony analysis revealed the existence of four clades among the L. longipalpis populations studied: (1) Laran, (2) Brazilian, (3) cis-Andean and (4) trans-Andean. We suggest that these clades represent species. A biogeographical interpretation of the molecular phylogeny suggests that the process of speciation in the L. longipalpis complex began in the Pliocene, from a sub-Andean-Amazonian gene pool resulting from the Andean orogeny (formation of the East Andean Cordillera). The four clades probably diverged as a result of vicariance events that occurred throughout the late Pliocene and Pleistocene. We propose and discuss several historical scenarios, based on the biogeography and historical geology of Central and South America.     

Lovesongs and period gene polymorphisms indicate Lutzomyia cruzi (Mangabeira, 1938) as a sibling species of the Lutzomyia longipalpis (Lutz and Neiva, 1912) complex

The sand fly Lutzomyia cruzi (Mangabeira, 1938) is implicated as a vector of American visceral leishmaniasis (AVL) in some areas of Brazil. Lutzomyia cruzi is closely related to Lutzomyia longipalpis s.l. (Lutz and Neiva, 1912) the main Latin American vector of AVL and a species complex. Although females of the two species are identical, the males can be distinguished by differences in the genitalia. Nevertheless, pheromone analysis shows that Lu. cruzi males produce 9-methyl-germacrene-B, which has also been found in a number of Latin American populations of Lu. longipalpis s.l. In addition, analysis of microsatellite loci shows that the level of divergence between Lu. cruzi and Lu. longipalpis s.l. is similar to that observed among the Lu. longipalpis s.l. sibling species. Here we present the lovesongs of Lu. cruzi males which are similar to the Burst-type songs produced by one of the Lu. longipalpis s.l. sibling species. We also present data on the molecular polymorphisms of the period gene of Lu. cruzi that indicate this species as another sibling within the Lu. longipalpis complex. The results highlight the importance of an integrative approach to understand the patterns of genetic and phenotypic divergence among very closely related vector species.     

Leishmaniasis in Bolivia. I. Lutzomyia longipalpis (Lutz & Neiva, 1912) as the vector of visceral leishmaniasis in Los Yungas

A relatively high leishmanial infection rate was found in the phlebotomine sandfly Lutzomyia longipalpis collected from three villages of the Los Yungas region (Department of La Paz, Bolivia). 2,578 female sandflies were dissected. In three houses surveyed in Santa Barbara promastigote infection rates of Lu. longipalpis were 4.2, 2.2 and 3.2% respectively. Anatomical localization of the infection in the insect, and biochemical characterization of the strains indicate that the parasite belongs to the Leishmania donovani complex. The geographical area and the biotopes of Lu. longipalpis are discussed in relation to the vector-parasite relationship.     

The acetylcholinesterase gene Ace: a diagnostic marker for the Pipiens and Quinquefasciatus forms of the Culex pipiens complex

The taxonomy of the Culex pipiens complex remains a controversial issue in mosquito systematics. Based on morphologic characters, 2 allopatric taxa are recognized, namely Cx. pipiens (including the form "molestus") in temperate areas and Cx. quinquefasciatus in tropical areas. Here we report on variability at the nucleotide level of an acetylcholinesterase gene in several strains and natural populations of this species complex. Few polymorphisms were found in coding regions within a subspecies but many polymorphisms were observed between subspecies in noncoding regions. We describe a method based on a restriction enzyme polymorphism in polymerase chain reaction-amplified DNA, in which the presence or absence of one restriction site discriminates Cx. pipiens, Cx. quinquefasciatus, and their hybrids. This technique reliably discriminates mosquitoes from more than 30 worldwide strains or populations. Polymerase chain reaction amplification of specific alleles may also be a useful tool for characterizing specific alleles of each sibling taxon.     

Molecular homogeneity in diverse geographical populations of Phlebotomus papatasi (Diptera, Psychodidae) inferred from ND4 mtDNA and ITS2 rDNA Epidemiological consequences.

An intraspecific study on Phlebotomus papatasi, the main proven vector of Leishmania major among the members of the subgenus Phlebotomus, was performed. The internal transcribed spacer 2 (ITS 2) of rDNA and the ND4 gene of mt DNA were sequenced from 26 populations from 18 countries (Albania, Algeria, Cyprus, Egypt, Greece, India, Iran, Israel, Italy, Lebanon, Morocco, Saudi Arabia, Spain, Syria, Tunisia, Turkey, Yugoslavia and Yemen), and compared. Samples also included three other species belonging to the subgenus Phlebotomus: P. duboscqi, a proven vector of L. major in the south of Sahara (three populations from Burkina Faso, Kenya and Senegal), P. bergeroti, a suspected vector of L. major (three populations from Oman Sultanate, Iran and Egypt), and one population of P. salehi from Iran. A phylogenetic study was carried out on the subgenus Phlebotomus. Our results confirm the validity of the morphologically characterized taxa. The position of P. salehi is doubtful. Variability in P. papatasi contrasts with that observed within other species having a wide distribution like P. (Paraphlebotomus) sergenti in the Old World or Lutzomyia (Lutzomyia) longipalpis in the New World. Consequently, it could be hypothesized that all populations of P. papatasi over its distribution area have similar vectorial capacities. The limits of the distribution area of L. major are correlated with the distribution of common rodents acting as hosts of the parasites.     

Copulation songs in three siblings of Lutzomyia longipalpis (Diptera: Psychodidae)

We present the results of recording male courtship songs of the sandfly Lutzomyia longipalpis. The striking differences in the songs from 3 Brazilian populations of this sandfly with 3 distinct male pheromones support the 3 sibling species previously proposed based on this characteristic.     

Genetic relationships among populations of Aedes aegypti in Taiwan by using phenotypic and random amplified DNA-polymerase chain reaction markers

An analysis of gene flow was conducted among collections of Aedes aegypti from 7 localities along the southwestern and southeastern coasts in Taiwan. Markers include 7 types of scaling patterns and 23 random amplified polymorphic DNA (RAPD) loci amplified by the polymerase chain reaction. Differences in scaling pattern and in the frequencies of RAPD markers were detected among populations and cluster analyses revealed 2 main groups on each side of the Central Mountain Range. Regression analysis of geographic distances and pairwise F(ST) values estimated from RAPD markers showed that southwestern populations are isolated by distance and that populations within 15 km are panmictic. This is a shorter distance than detected among collections of Ae. aegypti in similar published studies from Mexico and Argentina.     

Antibodies against Lutzomyia longipalpis saliva in the fox Cerdocyon thous and the sylvatic cycle of Leishmania chagasi

Sera of 11 wild Cerdocyon thous foxes from an endemic area for American visceral leishmaniasis were tested for the presence of antibodies against salivary gland homogenates (SGH) of Lutzomyia longipalpis. All foxes had higher levels of anti-Lu. longipalpis SGH antibodies than foxes from non-endemic areas, suggesting contact between foxes and the vector of visceral leishmaniasis. Sera of humans and dogs living in the same area were also tested for reactivity against Lu. longipalpis SGHs and had a lower proportion of reactivity than foxes. Antibodies against Leishmania chagasi were not detected in any of the foxes, but three foxes showed the presence of parasites in the bone marrow by direct examination, PCR or by infecting the vector. Both humans and dogs had higher levels of anti-Le. chagasi IgG antibodies than C. thous. The finding of an antibody response against saliva of Lu. longipalpis among C. thous together with the broad distribution of the vector in resting areas of infected foxes suggests that the natural foci of transmission of Le. chagasi exists independently of the transmission among dogs and humans.     

Micro- and minisatellite-expressed sequence tag (EST) markers discriminate between populations of Rhipicephalus appendiculatus

Biological differences, including vector competence for the protozoan parasite Theileria parva have been reported among populations of Rhipicephalus appendiculatus (Acari: Ixodidae) from different geographic regions. However, the genetic diversity and population structure of this important tick vector remain unknown due to the absence of appropriate genetic markers. Here, we describe the development and evaluation of a panel of EST micro- and minisatellite markers to characterize the genetic diversity within and between populations of R. appendiculatus and other rhipicephaline species. Sixty-six micro- and minisatellite markers were identified through analysis of the R. appendiculatus Gene Index (RaGI) EST database and selected bacterial artificial chromosome (BAC) sequences. These were used to genotype 979 individual ticks from 10 field populations, 10 laboratory-bred stocks, and 5 additional Rhipicephalus species. Twenty-nine markers were polymorphic and therefore informative for genetic studies while 6 were monomorphic. Primers designed from the remaining 31 loci did not reliably generate amplicons. The 29 polymorphic markers discriminated populations of R. appendiculatus and also 4 other Rhipicephalus species, but not R. zambeziensis. The percentage Principal Component Analysis (PCA) implemented using Multiple Co-inertia Analysis (MCoA) clustered populations of R. appendiculatus into 2 groups. Individual markers however differed in their ability to generate the reference typology using the MCoA approach. This indicates that different panels of markers may be required for different applications. The 29 informative polymorphic micro- and minisatellite markers are the first available tools for the analysis of the phylogeography and population genetics of R. appendiculatus.     

Sandflies (Diptera, Psychodidae) from an endemic leishmaniasis area in the cerrado region of the State of Maranh?o, Brazil]

This article presents a list of ten sandfly species from the genus Lutzomyia Fran?a, 1924 found in the counties of Aldeias Altas, Capinzal do Norte, Caxias, Codó, Coelho Neto, Timbiras, Timon and Tuntum in northeastern Maranh?o, Brazil. Presence of sandflies was associated with cases of visceral and cutaneous leishmaniasis. Some 377 specimens were captured indoors with CDC light traps and 1491 specimens in the peridomicile. The species were: Lutzomyia cortelezii, Lutzomyia evandroi, Lutzomyia goiana, Lutzomyia intermedia, Lutzomyia lenti, Lutzomyia longipalpis, Lutzomyia longipennis, Lutzomyia squamiventris, Lutzomyia termitophila and Lutzomyia whitmani. The most abundant species was L. longipalpis (67. 4% and 70.2%) followed by L. whitmani (31.0% and 24.7%). L. longipalpis was captured both indoors and outdoors in all the months studied.     

Effects of environmental pollution on microsatellite DNA diversity in wood mouse (Apodemus sylvaticus) populations

Ten microsatellite DNA loci were surveyed to investigate the effects of heavy metal pollution on the genetic diversity and population genetic structure of seven wood mouse (Apodemus sylvaticus) populations along a heavy metal pollution gradient away from a nonferrous smelter in the south of Antwerp (Flanders, Belgium). Analysis of soil heavy metal concentrations showed that soil Ag, As, Cd, Cu, and Pb decreased with increasing distance from the smelter. Genetic analyses revealed high levels of genetic variation in all populations, but populations from the most polluted sites in the gradient did not differ from those of less-polluted sites in terms of mean observed and expected heterozygosity level and mean allelic richness. No correlation was found between measures of genetic diversity and the degree of heavy metal pollution. However, an analysis of molecular variance and a neighbor-joining tree suggested a contamination-related pattern of genetic structuring between the most polluted and less polluted sites. Pairwise F(ST) values indicated that populations were significantly genetically differentiated, and assignment tests and direct estimates of recent migration rates suggested restricted gene flow among populations. Additionally, genetic differentiation increased significantly with geographical distance, which is consistent with an isolation-by-distance model. We conclude that, at least for our microsatellite DNA markers, genetic diversity in the studied wood mouse populations is not affected greatly by the heavy metal pollution.     

Leishmaniasis in Brazil. XIX: visceral leishmaniasis in the Amazon Region, and the presence of Lutzomyia longipalpis on the Island of Marajó, Pará State

Sporadic cases of visceral leishmaniasis in Amazonian Brazil appear limited to Pará State, in the lower Amazon valley and principally near the Atlantic coast. The fox Cerdocyon thous (L.) has been incriminated as a natural host of the causative parasite, Leishmania donovani chagasi, but past doubts have existed over the identification of the most likely vector as Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva, 1912). Investigations on two of five recent cases of visceral leishmaniasis of man in the Districts of Cachoeira do Arari and Salvaterra, on the eastern part of the Island of Marajó, Pará showed undoubted Lu. longipalpis to be abundant in one house and in numerous chicken-houses. This is the first record of Lu. longipalpis on Marajó Island, and the finding supports previous implication of this sandfly in the epidemiology of visceral leishmaniasis in other parts of Pará. Morphological differences have been noted between this insect from Marajó and other specimens from more highly endemic regions in the States of Ceará and Minas Gerais, Brazil.     

Morphometric and molecular characterization of the series Guyanensis (Diptera, Psychodidae, Psychodopygus) from the Ecuadorian Amazon Basin with description of a new species

Several populations of the series Guyanensis (Diptera, Psychodidae, Psychodopygus) were collected from the Yasuní National Park in the Ecuadorian Amazon region. The specimens comprised the species Psychodopygus geniculatus, Psychodopygus luisleoni and Psychodopygus corossoniensis. Within Ps. geniculatus, we observed two populations, one with a narrow paramere and relatively short genital filaments and the other characterized by a wider coxite and longer genital filaments. A multiple approach combining morphology, morphometry and DNA sequencing of the ribosomal internal transcribed spacer 2 (ITS2) and the mitochondrial cytochrome b gene was carried out. Morphological, morphometric and molecular data strongly suggested the presence of two populations within Ps. geniculatus. The lack of intermediate forms within these populations supported the proposal of two sympatric species. This report describes Psychodopygus francoisleponti n. sp.     

Lutzomyia longipalpis naturally infected by Leishmania (L.) chagasi in Várzea Grande, Mato Grosso State, Brazil, an area of intense transmission of visceral leishmaniasis

The American visceral leishmaniasis (AVL) is caused by parasites belonging to the genus Leishmania (Trypanosomatidae) and is transmitted to humans through the bite of certain species of infected phlebotomine sand flies. In this study, we investigated the natural infection ratio of Lutzomyia longipalpis, the main vector species of AVL in Brazil, in Várzea Grande, Mato Grosso State. Between July 2004 and June 2006, phlebotomine sand flies were captured in peridomestic areas using CDC light-traps. Four hundred and twenty (420) specimens of Lu. longipalpis were captured. 42 pools, containing 10 specimens of Lu. longipalpis each, were used for genomic DNA extraction and PCR (polymerase chain reaction) amplification. Leishmania spp. DNA was detected in three out of the 42 pools tested, resulting in a minimal infection ratio of 0.71%. Restriction fragment length polymorphism (RFLP) analysis indicated that Leishmania (L.) chagasi was the infective agent in the positive pools.     

Natural infection of Lutzomyia longipalpis by Leishmania sp. in Teresina, Piauí State, Brazil

In Brazil, control of the vector Lutzomyia longipalpis is one of the main strategies used to limit the expansion of American visceral leishmaniasis. However, studies on the ecology of this sand fly are rare, especially regarding its natural infection with species of Leishmania. A study of the natural infection of Lu. longipalpis by Leishmania sp. was carried out in the Bela Vista neighborhood in the city of Teresina, Piauí State, Brazil, an important area of American visceral leishmaniasis transmission. From February 2004 to January 2005, sand flies were captured with CDC light traps. Approximately 10 female sand flies in each capture were dissected and examined for the presence of evolutionary forms of Leishmania sp. Two sand fly species were identified: 1,832 were Lu. longipalpis and six Lu. whitmani. Twenty female sand flies (1.1%), all Lu. longipalpis, were infected with procyclic and nectomonad forms of Leishmania sp., found mostly in the hindgut. Higher proportions of infected sand flies were found four months after the rainy season, suggesting that environmental factors may predict not only vector abundance (as already known) but also their level of infection.     

Leishmaniasis in Brazil. XXI. Visceral leishmaniasis in the Amazon Region and further observations on the role of Lutzomyia longipalpis (Lutz & Neiva, 1912) as the vector

Further evidence is presented incriminating the sandfly Lutzomyia longipalpis as the vector of Leishmania chagasi, the causative agent of American visceral leishmaniasis, in the Amazon Region of Brazil. During an outbreak of the disease in Santarém, Pará State, this insect was shown to be the only species of sandfly consistently present in and around the patient's homes, where it often occurred in very large numbers. Of 491 specimens dissected, 35 (7.14%) proved to be infected, and isolates of L. chagasi were made from 16 of 27 of these sandflies following the inoculation of the promastigotes into hamsters. Finally, the parasite was transmitted to four other hamsters which had been subjected to the bites of large numbers of wild-caught Lu. longipalpis. Isolates of Leishmania from Lu. longipalpis captures in Santarém, and in another focus of visceral leishmaniasis on the Island of Marajó, Pará, have been shown to be biologically and biochemically indistinguishable from the parasite infecting man, dogs and foxes in Pará, and from stocks obtained from man elsewhere in Brazil (Bahia and Ceará States).     

Genetic characterization of a new variant within the ET-37 complex of Neisseria meningitidis associated with outbreaks in various parts of the world

A new variant within the electrophoretic type (ET)-37 complex of Neisseria meningitidis, ET-15, first detected in Canada in 1986, has been associated with severe clinical infections and high mortality rates in several European countries, Israel and Australia. To ascertain the genetic and epidemiological relationships of ET-15 strains from different geographical areas, 72 ET-15 isolates from 10 countries were compared to 13 isolates representing other clones of the ET-37 complex. The 85 strains were analysed by pulsed-field gel electrophoresis (PFGE) using 2 restriction endonucleases and Southern hybridization with 10 genetic markers. Four ET-15 strains and 4 other strains of the ET-37 complex were further examined using an additional restriction enzyme and a total of 18 genetic markers. PFGE fingerprints of the ET-15 strains were closely related. Strains within each country were even more closely related, suggesting single introductions of the clone. Physical mapping of genes in ET-15 and other strains of the ET-37 complex demonstrated that large genetic rearrangements of the genome have occurred in association with the appearance of the ET-15 variant.     

Practical population group assignment with selected informative markers: characteristics and properties of Bayesian clustering via STRUCTURE

Population stratification, which is caused by population genetic substructure (PGS), is a critical issue for the design and interpretation of genetic association studies. Methods to address this problem have been devised, but little is known at this point about practical genotyping requirements for resolving PGS based on different marker characteristics. In this report, we seek to (1) identify a small, practical marker set to differentiate African Americans (AAs) from European Americans (EAs), and (2) assess the impact of marker efficiency and sample size on clustering individuals into subgroups by the methods of STRUCTURE (Pritchard et al., [2000a] Genetics 155:945-959). A panel of 37 markers was genotyped for 865 individuals (640 EAs and 225 AAs) from the Northeastern United States. Among EAs, the assignment accuracy reached >99% using only the 4 most efficient markers. Among AAs, the assignment accuracy exceeded 95% when using the 6 most informative markers. Smaller sample size increased the variance in population differentiation, rather than degrading the results consistently. We conclude that the use of marker-efficiency measures for marker selection yielded a relatively small set of STR markers that were effective at differentiating EA and AA populations. The number of markers required is much lower than has been suggested in previous studies.     

Trypanosoma brucei gambiense Type 1 populations from human patients are clonal and display geographical genetic differentiation

We have rigorously tested the hypothesis that Trypanosoma brucei gambiense Type 1 is composed of genetically homogenous populations by examining the parasite population present in Human African Trypanosomiasis (HAT) patients from the Democratic Republic of Congo (DRC) and Cameroon (CAM). We amplified eight microsatellite markers by PCR directly from blood spots on FTA filters, thereby avoiding the significant parasite selection inherent in the traditional isolation techniques of rodent inoculation or in vitro culture. All microsatellite markers were polymorphic, although for four markers there was only polymorphism between the DRC and CAM populations, not within populations, suggesting very limited genetic exchange. Within the largest population from the DRC, Hardy–Weinberg equilibrium is not evident at any loci. This evidence suggests a clonal population. However, there was significant sub-structuring between the DRC and CAM samples (F_ST = 0.32), indicating that Trypanosoma brucei gambiense Type 1 has genetically distinct clades. The data combine to indicate that genetic exchange plays a very limited role. The finding of distinct clades in different places suggests the possibility that samples from humans with clinical signs represent clonal expansions from an underlying population that requires identifying and characterising.