A genotyping array for the globally invasive vector mosquito, Aedes albopictus.

BACKGROUND: Although whole-genome sequencing (WGS) is the preferred genotyping method for most genomic analyses, limitations are often experienced when studying genomes characterized by a high percentage of repetitive elements, high linkage, and recombination deserts. The Asian tiger mosquito (Aedes albopictus), for example, has a genome comprising up to 72% repetitive elements, and therefore we set out to develop a single-nucleotide polymorphism (SNP) chip to be more cost-effective. Aedes albopictus is an invasive species originating from Southeast Asia that has recently spread around the world and is a vector for many human diseases. Developing an accessible genotyping platform is essential in advancing biological control methods and understanding the population dynamics of this pest species, with significant implications for public health. METHODS: We designed a SNP chip for Ae. albopictus (Aealbo chip) based on approximately 2.7 million SNPs identified using WGS data from 819 worldwide samples. We validated the chip using laboratory single-pair crosses, comparing technical replicates, and comparing genotypes of samples genotyped by WGS and the SNP chip. We then used the chip for a population genomic analysis of 237 samples from 28 sites in the native range to evaluate its usefulness in describing patterns of genomic variation and tracing the origins of invasions. RESULTS: Probes on the Aealbo chip targeted 175,396 SNPs in coding and non-coding regions across all three chromosomes, with a density of 102 SNPs per 1 Mb window, and at least one SNP in each of the 17,461 protein-coding genes. Overall, 70% of the probes captured the genetic variation. Segregation analysis found that 98% of the SNPs followed expectations of single-copy Mendelian genes. Comparisons with WGS indicated that sites with genotype disagreements were mostly heterozygotes at loci with WGS read depth < 20, while there was near complete agreement with WGS read depths > 20, indicating that the chip more accurately detects heterozygotes than low-coverage WGS. Sample sizes did not affect the accuracy of the SNP chip genotype calls. Ancestry analyses identified four to five genetic clusters in the native range with various levels of admixture. CONCLUSIONS: The Aealbo chip is highly accurate, is concordant with genotypes from WGS with high sequence coverage, and may be more accurate than low-coverage WGS.

The population structure of Lutzomyia verrucarum (Diptera: Psycodidae), a Bartonella bacilliformis and Leishmania peruviana vector in Peru.

The population genetic structure of Lutzomyia verrucarum (Townsend), a sand fly disease vector of Carrion's disease and cutaneous leishmaniasis in the Peruvian Andes, was characterized by sequencing 653 bp of cytochrome b and 1,125 bp of the NADH dehydrogenase subunit 4 genes of its mitochondrial genome. DNA sequence variation within and between valleys was compared in a sample of 220 sand flies from three valleys (Purisima, Huaylas, and Conchucos) and five departments (Amazonas, Cajamarca, Piura, Lima, and Huancavelica). Gene network and phylogenetic analyses indicated a high similarity of haplotypes collected within a single valley (0-0.52% nucleotide divergence). Flies from each valley had unique genotypes not shared with specimens from other valleys or from more distant regions (0.8-3.1% nucleotide divergence). Mountain ranges and geographic distance appear to have impeded migration (N(m) = < 0.18) between valleys and separated populations into discrete genetic units.

Phlebotomine vector ecology in the domestic transmission of American cutaneous leishmaniasis in Chaparral, Colombia.

Phlebotomine vector ecology was studied in the largest recorded outbreak of American cutaneous leishmaniasis in Colombia in 2004. In two rural townships that had experienced contrasting patterns of case incidence, this study evaluated phlebotomine species composition, seasonal abundance, nocturnal activity, blood source, prevalence of Leishmania infection, and species identification. CDC miniature light traps were used to trap the phlebotomines. Traps were set indoors, peridomestically, and in woodlands. Natural infection was determined in pools by polymerase chain reaction-Southern blot, and blood sources and species identification were determined by sequencing. Large differences were observed in population abundance between the two townships evaluated. Lutzomyia longiflocosa was the most abundant species (83.1%). Abundance was higher during months with lower precipitation. Nocturnal activity was associated with human domestic activity. Blood sources identified were mainly human (85%). A high prevalence of infection was found in L. longiflocosa indoors (2.7%) and the peridomestic setting (2.5%). L. longiflocosa was responsible for domestic transmission in Chaparral.

Morphological variants of Aedes aegypti collected from the Leeward Island of Antigua.

Nineteen Aedes aegypti larvae were collected in rural Antigua, West Indies, from an 18-liter plastic bucket. The location was in a rural area at the northern end of Antigua bordering the coast of Dickenson Bay and approximately 50 m south of Halcyon Cove Beach (17 degrees 09'42.54"N, 61 degrees 50'44.50"W; elevation 16 m). Atypical morphology was noted in larvae and 3 reared adult females. Fourth instars showed a reduction in length of the lateral hair on the saddle (seta 1-X) with measurements ranging from 0.36 to 0.57 the length of the saddle. Two atypical female specimens displayed an abundance of dull white to gold scales that blanketed the abdomen. A 3rd specimen bore fine, golden scales on the mesonotum and bronze scales on the vertices of the head. These adult specimens demonstrated morphological characteristics that closely parallel described mutations, although the genetic basis for these characters was not confirmed. The remaining adults in the collection were morphologically typical. Adults and larvae were compared to field populations from Florida, Bahamas, and Antigua, as well as to the Rockefeller strain maintained at Rutgers University.

Phylogenetics of the phlebotomine sand fly group Verrucarum (Diptera: Psychodidae: Lutzomyia).

Within the sand fly genus Lutzomyia, the Verrucarum species group contains several of the principal vectors of American cutaneous leishmaniasis and human bartonellosis in the Andean region of South America. The group encompasses 40 species for which the taxonomic status, phylogenetic relationships, and role of each species in disease transmission remain unresolved. Mitochondrial cytochrome c oxidase I (COI) phylogenetic analysis of a 667-bp fragment supported the morphological classification of the Verrucarum group into series. Genetic sequences from seven species were grouped in well-supported monophyletic lineages. Four species, however, clustered in two paraphyletic lineages that indicate conspecificity--the Lutzomyia longiflocosa-Lutzomyia sauroida pair and the Lutzomyia quasitownsendi-Lutzomyia torvida pair. COI sequences were also evaluated as a taxonomic tool based on interspecific genetic variability within the Verrucarum group and the intraspecific variability of one of its members, Lutzomyia verrucarum, across its known distribution.

Leishmania (Viannia) infection in the domestic dog in Chaparral, Colombia.

Peridomestic transmission of American cutaneous leishmaniasis is increasingly reported and dogs may be a reservoir of Leishmania (Viannia) in this setting. We investigated the prevalence of infection in dogs in Chaparral County, Colombia, the focus of an epidemic of human cutaneous leishmaniasis caused by Leishmania (Viannia) guyanensis. Two (0.72%) of 279 dogs had lesions typical of cutaneous leishmaniasis that were biopsy positive by kinetoplast DNA polymerase chain reaction-Southern blotting. Seroprevalence was 2.2% (6 of 279) by enzyme-linked immunosorbent assay. Buffy coat and ear skin biopsy specimens were positive by polymerase chain reaction-Southern blotting in 7.3% (10 of 137) and 11.4% (12 of 105) of dogs, respectively. Overall 20% of dogs (21 of 105) showed positive results for one or more tests. Amplification and sequencing of the Leishmania 7SL RNA gene identified L. guyanensis in one dog and L. braziliensis in two dogs. No association was identified between the risk factors evaluated and canine infection. Dogs may contribute to transmission but their role in this focus appears to be limited.

Environmental risk factors for the incidence of American cutaneous leishmaniasis in a sub-Andean zone of Colombia (Chaparral, Tolima).

Environmental risk factors for cutaneous leishmaniasis were investigated for the largest outbreak recorded in Colombia. The outbreak began in 2003 in Chaparral, and in the following five years produced 2,313 cases in a population of 56,228. Candidate predictor variables were land use, elevation, and climatic variables such as mean temperature and precipitation. Spatial analysis showed that incidence of cutaneous leishmaniasis was higher in townships with mean temperatures in the middle of the county's range. Incidence was independently associated with higher coverage with forest or shrubs (2.6% greater for each additional percent coverage, 95% credible interval [CI] = 0.5-4.9%), and lower population density (22% lower for each additional 100 persons/km(2), 95% CI = 7-41%). The extent of forest or shrub coverage did not show major changes over time. These findings confirmed the roles of climate and land use in leishmaniasis transmission. However, environmental variables were not sufficient to explain the spatial variation in incidence.

NEW RECORDS OF PHLEBOTOMINE SAND FLIES (DIPTERA: PSYCHODIDAE) FROM ECUADOR.

The number of recorded phlebotomine sand fly species in Ecuador has nearly doubled during the past 20 years as a result of surveys. In 2005, a sand fly survey of two localities, Tiputini in the Amazon rain forest and Paraiso Escondido in the Pacific coastal lowland forest, resulted in the capture of 25 species. New records for Ecuador consisted of five species from the Amazonian region and one from Paraiso Escondido. The Amazonian species were Nyssomyia richardwardi (Ready and Fraiha), Psathyromyia dreisbachi (Causey and Damasceno), Psathyromyia runoides (Fairchild and Hertig), Trichophoromyia pabloi (Barretto, Burbano and Young), and Trichopygomyia witoto (Young and Morales). The Pacific coastal lowland species was Psathyromyia punctigeniculata (Floch and Abonnenc).

Population Genetics of Aedes albopictus (Diptera: Culicidae) Invading Populations, Using Mitochondrial nicotinamide Adenine Dinucleotide Dehydrogenase Subunit 5 Sequences.

Aedes albopictus (Skuse) (Diptera: Culicidae), the Asian tiger mosquito indigenous to Asia, now an invasive species worldwide, is an important vector for several arboviruses. Genetic analysis using the mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 5 (ND5) gene was carried out in populations from Cameroon (n = 50), Hawaii (n = 38), Italy (n = 20), the continental United States, Brazil, and its native range. Data for Brazil, the continental United States, and the native range was obtained from Birungi and Munstermann (2002). Direct sequencing was used to identity unique haplotypes. The limited phylogeographic partitioning of haplotypes with low levels of sequence divergence in both Cameroon and Hawaii was consistent with the population structure of Ae. albopictus in the United States and Brazil. Four new haplotypes were identified from the samples from Cameroon and Hawaii, adding to previously described haplotypes. Hawaii shared a haplotype with Cameroon that was unique to these two regions. Hawaii also had higher overall haplotype diversity than seen in previous continental United States, Brazil, or native range populations. Hawaiian, Cameroon, and Italian populations did not share haplotypes with Brazil, which validates the earlier mitochondrial DNA studies indicating a separate introduction of this species into Brazil.

Light-emitting diode technology improves insect trapping.

In a climate of increased funding for vaccines, chemotherapy, and prevention of vector-borne diseases, fewer resources have been directed toward improving disease and vector surveillance. Recently developed light-emitting diode (LED) technology was applied to standard insect-vector traps to produce a more effective lighting system. This approach improved phlebotomine sand fly capture rates by 50%, and simultaneously reduced the energy consumption by 50-60%. The LEDs were incorporated into 2 lighting designs, 1) a LED combination bulb for current light traps and 2) a chip-based LED design for a modified Centers for Disease Control and Prevention light trap. Detailed descriptions of the 2 designs are presented.

Distribución de los vectores de Leishmania infantum (Kinetoplastida: Trypanosomatidae) en Colombia / Distribution of Leishmania infantum vector species in Colombia

Introducción. Debido a la importancia que tiene la vigilancia entomológica como principal medida de control en el manejo de la leishmaniasis visceral, es necesario contar con información actualizada acerca de la distribución y ecología de los insectos involucrados en la transmisión para optimizar las estrategias de prevención. Objetivo. Presentar la distribución actualizada geo-referenciada de L. longipalpis y L. evansi, vectores de los parásitos que causan leishmaniasis visceral en Colombia, teniendo en cuenta la asociación de los insectos con su hábitat. Materiales y métodos. Los registros de distribución se obtuvieron a partir de los ejemplares recolectados en Colombia desde 1967. La información obtenida se organizó en una base de datos a partir de la cual se tomaron las localidades que, posteriormente, fueron sometidas a análisis geográficos por medio de Arc View que se utilizaron para realizar los mapas de distribución. Resultados. Para L. longipalpis se obtuvieron 40 localidades todas distribuidas a lo largo del valle del río Magdalena: Alto (24), Medio (11) y Bajo (5) Magdalena. L. evansi fue registrado en 19 localidades también ubicadas en el mismo valle: cinco en el Magdalena Medio y 14 el Magdalena Bajo. Conclusiones. Ambas especies demostraron una consistente asociación con regiones clasificadas principalmente como bosque seco tropical según las zonas de vida de Holdridge lo que confirma el riesgo epidemiológico de leishmaniasis visceral en estas áreas

Introduction. Since entomological surveillance is the main control strategy for visceral leishmaniasis, updated information on the distribution and ecology of involved vector species is necessary for planning preventive measures. Objective. To present the updated and geo-referenced distribution of L. longipalpis and L. evansi, vectors of visceral leishmaniasis in Colombia, considering their relationship with their habitat. Materials and methods. Distribution was estimated from records of the sand fly specimens collected since 1967.The information was organized in a database from which the localities were selected and geographically analyzed with Arc view in order to develop the distribution maps. Results. 40 localities were established for L. longipalpis along the upper (24), middle (11) and lower (5) Magdalena river valley . L. evansi was recorded in 19 localities of the middle (5) and lower (14) Magdalena valley. Conclusions. Both species showed consistent association with dry tropical forest ( sensu Holdridge 1967), confirming the epidemiological risk for visceral leishmaniasis in these areas.

[Distribution of Leishmania infantum vector species in Colombia]. / Distribución de los vectores de Leishmania infantum (Kinetoplastida: Trypanosomatidae) en Colombia.

INTRODUCTION: Since entomological surveillance is the main control strategy for visceral leishmaniasis, updated information on the distribution and ecology of involved vector species is necessary for planning preventive measures. OBJECTIVE: To present the updated and geo-referenced distribution of L. longipalpis and L. evansi, vectors of visceral leishmaniasis in Colombia, considering their relationship with their habitat. MATERIALS AND METHODS: Distribution was estimated from records of the sand fly specimens collected since 1967. The information was organized in a database from which the localities were selected and geographically analyzed with Arc view in order to develop the distribution maps. RESULTS: 40 localities were established for L. longipalpis along the upper (24), middle (11) and lower (5) Magdalena river valley. L. evansi was recorded in 19 localities of the middle (5) and lower (14) Magdalena valley. CONCLUSIONS: Both species showed consistent association with dry tropical forest (sensu Holdridge 1967), confirming the epidemiological risk for visceral leishmaniasis in these areas.

Systematic relationships among Lutzomyia sand flies (Diptera: Psychodidae) of Peru and Colombia based on the analysis of 12S and 28S ribosomal DNA sequences.

Lutzomyia spp. are New World phlebotomine sand flies, many of which are involved in the transmission of human diseases, such as leishmaniases and bartonellosis. The systematic classification of the approximately 400 species in the genus has been based on morphological characters, but the relationships within the genus are still very much in question. We have inferred phylogenies of 32 species of phlebotomine sand flies belonging to seven sub-genera and two species groups, by using fragments of the mitochondrial small subunit (12SrRNA) and of the nuclear large subunit (28SrRNA) ribosomal gene sequences. The subgenus Helcocyrtomyia and the Verrucarum species group, prominent representatives of the Peruvian sand fly fauna, were represented by 11 and 7 species, respectively. Although based on a limited number of taxa, the resulting phylogenies, based on 837 characters, provide an initial phylogenetic backbone for the progressive reconstruction of infrageneric relationships within Lutzomyia.

Biochemical taxonomy and enzyme electrophoretic profiles during development, for three morphologically similar Aedes species (Diptera: Culicidae) of the subgenus Stegomyia.

Aedes (Stegomyia) cretinus is a rarely documented mosquito with a Mediterranean distribution, whereas Aedes (S.) albopictus has spread worldwide in the past two decades because of its anthropogenic associations. A third closely related species, Aedes (S.) flavopictus, is sympatric with A. albopictus in northeast Asia. The three species are characterized by a striking mid-thoracic white stripe and, consequently, field-collected individuals may be difficult to separate by morphology. Sixteen biochemical markers were described for laboratory strains representing the three species; these provided the first biochemical genetic profile for A. cretinus and A. flavopictus. Diagnostic enzymes for identifying each species pair were determined. A biochemical key was provided to distinguish among adults of the three species. Several enzyme loci that were diagnostic for the adult stage proved unreliable for identifying immature stages. Voucher specimens for link-reared series of larva, pupa, adult male, and adult female stages of the A. cretinus Crete strain (n = 88) and the A. albopictus Nepal strain (n = 105) were deposited at the Yale Peabody Museum of Natural History, New Haven, CT.

Mitochondrial cytochrome b variation in populations of the visceral leishmaniasis vector Lutzomyia longipalpis across eastern Brazil.

A population analysis of peridomestic, light-trapped, field specimens of the phlebotomine sand fly Lutzomyia longipalpis was targeted to six locations representing a geographic transect across eastern Brazil. Mitochondrial cytochrome b gene sequences established the pattern of genetic variation among the populations. Alignment of a 261-basepair region at the 3' end of cytochrome b identified 30 haplotypes and 21 segregating sites from 78 sand flies. Pairwise comparisons indicated statistically significant population structuring between northern and southern populations, as well as structuring among the southern populations. Prominent spatial clustering was evident for two of the populations in a minimum spanning network of the haplotypes, but sequence divergence was not sufficient to indicate cryptic species.

Infection of New- and Old-World Aedes albopictus (Diptera: Culicidae) by the intracellular parasite Wolbachia: implications for host mitochondrial DNA evolution.

Wolbachia are cytoplasmically inherited, endosymbiotic bacteria known to infect a wide variety of arthropods. Polymerase chain reaction (PCR) amplification of the Wolbachia surface protein (wsp) gene was used to assay the infection of geographically disparate populations of Aedes albopictus (Skuse) by Wolbachia. Nine North American, four South American, one Hawaiian, and four Old World populations of A. albopictus were all doubly infected with both the wAlbA and wAlbB strains of Wolbachia. A 365-bp region of the wAlbA wsp gene was sequenced from seven geographically disparate host populations, and all sequences were identical. Similarly, a 474-bp region of the wAlbB wsp gene was sequenced from the same populations, and all sequences were identical. These results suggest a role for Wolbachia infection in causing the previously established pattern of low mitochondrial DNA variability, but average nuclear gene diversity, within and among populations of A. albopictus.

[PCR as a tool in confirming the experimental transmission of Leishmania chagasi to hamsters by Lutzomyia longipalpis (Diptera:Psychodidae)]. / PCR para la confirmación de transmisión experimental de Leishmania chagasi a hámster sano por picadura de Lutzomyia longipalpis (Diptera: Psychodidae).

The use of PCR (polymerase chain reaction) was evaluated for its effectiveness as a tool in the detection of transmission of Leishmania chagasi to a hamster host, Mesocricetus auratus, by insect vector bite. Two pairs of uninfected and anesthetized hamsters were introduced into cages containing infected females of the typical phlebotomine sand fly vector, Lutzomyia longipalpis. The flies were experimentally infected with Leishmania chagasi and the infection was verified by dissection of subsamples. At 37 and 51 days after exposure to the infected flies, biopsies of each hamster's liver and spleen were subjected to direct histopathological and PCR examination. DNA was extracted with Chelex 100; for PCR amplification, primers specific to Leishmania minicircle DNA were used. PCR product was separated on agarose gels and visualized with UV. A band of approximately 120 base pairs was observed in 3 of the 4 biopsies, corresponding to the expected minicircle size. PCR was the only method that detected presence of the parasite. The results demonstrated that the sensitivity of PCR greatly expedites the confirmation process of a particular phlebotomine species as a vector of leishmaniasis.

PCR para la confirmación de trasmisión experimental de Leishmania chagasi a hámster sano por picadura de Lutzomyia longipalpis (Diptera: Psychodidae) / PCR as a tool in confirming the experimental transmission of Leishmania chagasi to hamsters by Lutzomyia longipalpis (Diptera: Psychodidae)

Se evaluó la efectividad de la PCR como herramienta en la detección de la transmisión experimental de Leishmania chagasi a hámster, Mesocricetus auratus, por picadura del insecto vector. Dos pares de hámsteres sanos y anestesiados fueron colocados en jaulas que contenían hembras de Lutzomyia longipalpis. Previamente, las hembras se infectaron experimentalmente con Leishmania chagasi y la infección se confirmó por disección en una submuestra. A los 37 y 51 días después de la exposición a los insectos infectados, las biopsias de hígado y bazo de cada hámster se sometieron a examen directo al microscopio, histopatología y PCR. El ADN se extrajo con Chelex 100(c); en la amplificación se utilizó un par de iniciadores específicos para la región conservada de los minicírculos del ADN de Leishmania. El producto amplificado se separó en geles de agarosa y se visualizó bajo luz UV. En tres de las cuatro biopsias se observó una banda de 120 pares de bases, aproximadamente, correspondiente al tamaño esperado de la fracción del minicírculo. La técnica de PCR fue el único método que detectó la presencia del par sito. Estos resultados demostraron que la sensibilidad de la PCR acelera los procesos de incriminación vectorial de las especies vectoras de leishmaniasis.

[Approximation to the CDC method to determine insecticide susceptibility in leishmaniasis vectors]. / Aproximación al método CDC para determinar susceptibilidad a insecticidas en vectores de leishmaniasis.

The Centers for Disease Control (CDC, USA) has proposed a simplified method for the determinations of insecticide resistance in adult mosquitoes, using 250 ml Wheaton bottles containing measured dosages. Insects are transferred into the bottle for 1 hour and monitored for mortality at regular intervals. In standardizing the CDC method for use with phlebotomine sand flies, effects of the solvent without insecticide were evaluated. Two colonized sand fly vector species were used: Lutzomyia longipalpis (F50 and F54) and Lutzomyia serrana (F17). Groups of 10 to 24 unfed females 1-3 days old were transferred for 1 h to Wheaton bottles with the following pretreatment: (1) without additive, (2) 0.5 ml of acetone, or (3) 1.0 ml of acetone. Three to 5 replicates were undertaken for each condition and each species. In the control bottles, the insects rested quietly and after 1 h appeared normal. In bottles with 0.5 and 1.0 ml acetone, a repellent effect was observed in L. longipalpis and L. serrana within the first 10 min. A small proportion of the L. serrana became prostrate, but recovered quickly after removal from the bottle. Field test performed with Lutzomyia quasitownsendi produced results simialar to those of the L. serrana colony flies. The insecticide bioassays were performed with L. longipalpis (F60) flies. Females were exposed to three graded doses of lambdacyhalothrin (10, 50 and 100 micrograms/bottle), and mortality was recorded at five-minute intervals. Regression lines for the 3 concentrations were compared within the context of the CDC method. The advantages of the CDC method over the WHO protocols were four: lower cost, fewer insects required, an entire group of insects exposed to the same surface, and ease of field use.