The transmission of visceral leishmaniasis in the municipality of Guarujá, on the Coast of São Paulo state, Brazil

ABSTRACT OBJECTIVE To perform an entomological survey, evaluating the circulation of Leishmania spp. in sand flies captured from the new foci of visceral leishmaniasis (VL) in the coastal region of São Paulo state. METHODS Sand flies were captured from November 2016 to September 2018 using light traps of the Centers for Disease Control (CDC), in the neighborhood where VL cases were reported. Leishmania spp. circulation was evaluated by gut dissection and molecular analysis of the females captured. RESULTS Nyssomyia intermedia was the more frequent species (90,7%) within the 1,203 sand flies captured. We found no flagellates in dissected females, but two pools containing females of Ny. intermedia presented DNA of L. infantum. CONCLUSION Our results suggest that Ny. intermedia might be involved in the establishment of this new VL focus in Sao Paulo. However, before incriminating this species as a L. infantum vector, further studies should investigate other vectorial capacity parameters, including competence, survival, and feeding habits.

The gene expression of Leishmania infantum chagasi inside Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil

Leishmania infantum chagasi is the causative agent and Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the Americas. We investigated the expression of Leishmania genes within L. longipalpis after artificial infection. mRNAs from genes involved in sugar and amino acid metabolism were upregulated at times of high parasite proliferation inside the insect. mRNAs from genes involved in metacyclogenesis had higher expression in late stages of infection. Other modulated genes of interest were involved in immunomodulation, purine salvage pathway and protein recycling. These data reveal aspects of the adaptation of the parasite to the microenvironment of the vector gut and reflect the preparation for infection in the vertebrate.

Phylogenetic relationships of closely-related phlebotomine sand flies (Diptera: Psychodidae) of Nyssomyia genus and Lutzomyia subgenus

BACKGROUND The Nyssomyia genus and Lutzomyia subgenus include medical important species that are Latin American leishmaniases vectors. Little is known about the phylogenetic relationships of closely-related species in each of these taxonomic groups that are morphologically indistinguishable or differentiated by very subtle details. OBJECTIVES We inferred the phylogenetic relationships of closely-related species within both the Nyssomyia genus and the Lutzomyia subgenus using a cytochrome c oxidase subunit I (COI) fragment. METHODS The sampling was carried out from 11 Argentinean localities. For genetic analyses, we used GenBank sequences in addition to our sequences from Argentina. Kimura 2-parameter (K2P) genetic distance and nucleotide divergence (Da) was calculated between closely-related species of Nyssomyia genus, Lutzomyia subgenus and between clades of Lutzomyia longipalpis complex. FINDINGS The K2P and Da values within species of Nyssomyia genus and Lutzomyia subgenus were lower than the divergence detected between clades of Lu. longipalpis complex. The haplotype network analyses within Lutzomyia subgenus showed shared haplotypes between species, contrary to Nyssomyia genus with none haplotype shared. Bayesian inference within Nyssomyia genus presented structuring by species. MAIN CONCLUSIONS This study evidences the phylogenetic proximity among closely-related species within Nyssomyia genus and Lutzomyia subgenus. The COI sequences of Nyssomyia neivai derived from the present study are the first available in GenBank.

Lutzomyia longipalpis (Diptera: Psychodidae) Argentina-Bolivia border: new report and genetic diversity

American visceral leishmaniasis (AVL) has two main scenarios of transmission as follows: scattered cases in rural areas and urban outbreaks. Urban AVL is in active dispersion from the northeastern border of Argentina-Paraguay-Brazil to the South. The presence of Lutzomyia longipalpis was initially reported in urban environments in the northwestern border of the country. The presence of Lu. longipalpis, environmental variables associated with its distribution, and its genetic diversity were assessed in Salvador Mazza, Argentina, on the border with Bolivia. The genetic analysis showed high haplotype diversity, low nucleotide diversity, and low nucleotide polymorphism index. We discuss the hypothesis of an expanding urban population with introgressive hybridisation of older haplogroups found in their path in natural forest or rural environments, acquiring a new adaptability to urban environments, and the possibility of changes in vector capacity.

Molecular validation of anthropophilic Phlebotominae sandflies (Diptera: Psychodidae) in Central Panama

Six Phlebotominae sand fly species are incriminated as biological vectors of human pathogens in Panama, but molecular corroboration is still needed. We aim at confirming the identity of Phlebotominae species documented as anthropophilic in Panama. Adult sandflies were collected from August 2010 to February 2012 in Central Panama using CDC light traps. Species confirmation was accomplished through molecular barcodes and allied sequences from GenBank. A total of 53,366 sand fly specimens representing 18 species were collected. Five species were validated molecularly as single phylogenetic clusters, but Psychodopygus thula depicted two genetically divergent lineages, which may be indicative of cryptic speciation.

Especies de flebotomíneos (Diptera: Psychodidae) recolectados en reservas naturales de las regiones del Darién y del Pacífico en Colombia / Species of sand flies (Diptera: Psychodidae) collected from natural reserves in the Pacific and Darien regions of Colombia

Resumen Introducción. Los departamentos de Chocó y Antioquia en Colombia presentan condiciones climáticas y de vegetación que favorecen el establecimiento de especies de vectores del género Lutzomyia y la transmisión de Leishmania spp. a poblaciones humanas que ingresan a ambientes selváticos conservados. Objetivo. Reportar las especies de flebotomíneos presentes en tres reservas naturales de las regiones del Darién y del Pacífico en Colombia. Materiales y métodos. Los flebotomíneos se recolectaron en las reservas naturales El Aguacate (Acandí, Chocó), Nabugá (Bahía Solano, Chocó) y Tulenapa (Carepa, Antioquia). La recolección se hizo con trampas de luz CDC, mediante búsqueda activa en sitios de reposo y con trampas Shannon. La determinación taxonómica de especies se basó en las claves taxonómicas. En algunas especies de interés taxonómico, se evaluó el uso de secuencias parciales de la región 5' del gen COI. Resultados. Se recolectaron 611 flebotomíneos adultos: 531 en Acandí, 45 en Carepa y 35 en Bahía Solano. Se identificaron 17 especies del género Lutzomyia, tres del género Brumptomyia y una del género Warileya. Las distancias genéticas (K2P) y los soportes de agrupación (>99 %) en el dendrograma de neighbor joining correspondieron a la mayoría de unidades taxonómicas operacionales moleculares (Molecular Operational Taxonomic Units, MOTU) establecidas para el grupo Aragaoi y confirmaron claramente la identidad de Lu. coutinhoi. Conclusión. Se identificaron especies que tienen importancia en la transmisión de la leishmaniasis en Acandí, Bahía Solano y Carepa. Se confirmó la presencia de Lu. coutinhoi en Colombia.

Abstract Introduction: The departments of Chocó and Antioquia in Colombia show climatic and vegetation conditions favoring the establishment of vector species of the genus Lutzomyia and the transmission of Leishmania spp. to human populations entering conserved forest environments. Objective: To report the species of Phlebotomine sandflies present in three natural reserves in the Darien and Pacific regions of Colombia. Materials and methods: Sand flies were collected specifically in the natural reserves El Aguacate (Acandí, Chocó), Nabugá (Bahía Solano, Chocó) and Tulenapa (Carepa, Antioquia). Sand flies were collected with CDC light traps, active search in resting places and Shannon traps. The taxonomic determination of species was based on taxonomic keys. For some species of taxonomic interest, we evaluated the partial sequences of the 5' region of COI gene. Results: A total of 611 adult sand flies were collected: 531 in Acandí, 45 in Carepa and 35 in Bahía Solano. Seventeen species of the genus Lutzomyia, three of the genus Brumptomyia and one of the genus Warileya were identified. The genetic distances (K2P) and grouping supported (>99%) in the neighbor joining dendrogram were consistent for most established molecular operational taxonomic units (MOTU) of the Aragaoi group and clearly confirmed the identity of Lu. coutinhoi. Conclusion: Species that have importance in the transmission of leishmaniasis in Acandí, Bahía Solano and Carepa were identified. The presence of Lu. coutinhoi was confirmed and consolidated in Colombia.

The current status of the Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) species complex

Lutzomyia longipalpis s.l. is a complex of sibling species and is the principal vector of American visceral leishmaniasis. The present review summarises the diversity of efforts that have been undertaken to elucidate the number of unnamed species in this species complex and the phylogenetic relationships among them. A wide variety of evidence, including chemical, behavioral and molecular traits, suggests very recent speciation events and complex population structure in this group. Although significant advances have been achieved to date, differential vector capacity and the correlation between structure of parasite and vector populations have yet to be elucidated. Furthermore, increased knowledge about recent epidemiological changes, such as urbanisation, is essential for pursuing effective strategies for sandfly control in the New World.

Genetic variability of populations of Nyssomyia neivai in the Northern State of Paraná, Brazil

ABSTRACT The genetic study of sandfly populations needs to be further explored given the importance of these insects for public health. Were sequenced the NDH4 mitochondrial gene from populations of Nyssomyia neivai from Doutor Camargo, Lobato, Japira, and Porto Rico, municipalities in the State of Paraná, Brazil, to understand the genetic structure and gene flow. Eighty specimens of Ny. Neivai were sequenced, 20 from each municipality, and 269 base pairs were obtained. A total of 27 haplotypes and 28 polymorphic sites were found, along with a haplotypic diversity of 0.80696 and a nucleotide diversity of 0.00567. Haplotype H5, with 33 specimens, was the most common among the four populations. Only haplotypes H5 and H7 were present in all four populations. The population from Doutor Camargo showed the highest genetic diversity, and only this population shared haplotypes with those from the other municipalities. The highest number of haplotypes was sheared with Lobato which also had the highest number of unique haplotypes. This probably occurred because of constant anthropic changes that happened in the environment during the first half of the twentieth century, mainly after 1998. There was no significant correlation between genetic and geographical distances regarding these populations. However, the highest genetic and geographical distances, and the lowest gene flow were observed between Japira and Porto Rico. Geographical distance is a possible barrier between these municipalities through the blocking of haplotype sharing.

Phlebotomine sandfly (Diptera: Psychodidae) diversity and their Leishmania DNA in a hot spot of American Cutaneous Leishmaniasis human cases along the Brazilian border with Peru and Bolivia

In this study, we identified the phlebotomine sandfly vectors involved in the transmission of American Cutaneous Leishmaniasis (ACL) in Assis Brasil, Acre, Brazil, which is located on the Brazil-Peru-Bolivia frontier. The genotyping of Leishmania in phlebotomines was performed using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. A total of 6,850 sandflies comprising 67 species were captured by using CDC light traps in rural areas of the municipality. Three sandfly species were found in the state of Acre for the first time: Lutzomyia georgii, Lu. complexa and Lu. evangelistai. The predominant species was Lu. auraensis/Lu. ruifreitasi and Lu. davisi (total 59.27%). 32 of 368 pools were positive for the presence of Leishmania DNA (16 pools corresponding to Lu. davisi, and 16 corresponding to Lu. auraensis/Lu. ruifreitasi), with a minimal infection prevalence of 1.85% in Lu. davisi and 2.05% in Lu. auraensis/Lu. ruifreitasi. The Leishmania species found showed maximum identity with L. (Viannia) guyanensis and L. (V.) braziliensis in both phlebotomine species. Based on these results and similar scenarios previously described along the Brazil/Peru/Bolivia tri-border, the studied area must take into consideration the possibility of Lu. davisi and Lu. auraensis/Lu. ruifreitasi as probable vectors of ACL in this municipality.

Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

When population and evolutionary genetics met behaviour

In this review, we analyse the impact of a population and evolutionary genetics approach on the study of insect behaviour. Our attention is focused on the model organism Drosophila melanogaster and several other insect species. In particular, we explore the relationship between rhythmic behaviours and the molecular evolution of clock and ion channel genes.

Molecular analysis of an odorant-binding protein gene in two sympatric species of Lutzomyia longipalpis s.l.

Lutzomyia longipalpis s.l. is the main vector of American visceral leishmaniasis (AVL) and occurs as a species complex. DNA samples from two Brazilian sympatric species that differ in pheromone and courtship song production were used to analyse molecular polymorphisms in an odorant-binding protein ( obp29 ) gene. OBPs are proteins related to olfaction and are involved in activities fundamental to survival, such as foraging, mating and choice of oviposition site. In this study, the marker obp29 was found to be highly polymorphic in Lu. longipalpis s.l. , with no fixed differences observed between the two species. A pairwise fixation index test indicated a moderate level of genetic differentiation between the samples analysed.

Genética de populações do gene paralytic e análise multilocus de genes que controlam o som de corte no complexo Lutzomyia longipalpis (Diptera: Psychodidae) / Population genetics of the paralytic gene and multilocus analysis of ncourtship song genes in the lutzomyia longipalpis complex (diptera: psychodidae)

Lutzomyia longipalpis é o principal vetor da leishmaniose visceral americana (LVA), erepresenta um complexo de espécies crípticas. Trabalhos anteriores sugeriram a existência de quatroespécies com distribuições geográficas distintas: (1) espécie A- Lu. longipalpis sensu stricto – Brasil(Exceto Roraima), (2) Laran – espécie B = Lu. pseudolongipalpis (Venezuela), (3) cis-Andina – espécie C(Colômbia, noroeste da Venezuela e incluíram Roraima no Brasil) e (4) trans-Andina – espécie D(América Central). Além disso, experimentos com cruzamentos, análise dos sons de cópula, feromônios,microsatelites e genes que controlam o som de corte, sugeriram que as populações brasileiras deste vetorrepresentam diferentes espécies que podem ser divididas em dois grupos principais de acordo com o tipode som (Burst ou Pulsado) que machos produzem durante a cópula. No entanto, nenhuma diferençadiagnóstica foi observada entre esses dois grupos na maioria dos marcadores moleculares utilizados até omomento. Inicialmente analisamos a divergência molecular em um fragmento do gene paralytic, um locusenvolvido no controle dos sons de corte em Drosophila, entre diversas populações de Lu. longipalpis doBrasil produzindo sons do tipo Burst ou Pulsado. Comparamos também Lu. longipalpis com uma espéciemuito próxima, Lutzomyia cruzi, que produz sons do tipo Burst. Os resultados indicaram um númeromaior de diferenças fixas entre Lu. cruzi e as populações de Lu. longipalpis com som tipo Pulsado quecom aquelas produzindo som do tipo Burst. Os dados confirmaram que as diferentes espécies docomplexo Lu. longipalpis no Brasil podem ser divididas em dois grupos, um representando uma únicaespecie e o segundo grupo mais heterogêneo que provavelmente representa diversas espécies incipientes.Também fizemos uma análise macrogeográfica da divergência e fluxo gênico no gene paralytic entrepopulações de Lu. longipalpis das Américas do Sul e Central...

Lutzomyia longipalpis is the main vector of American Visceral Leishmaniasis, and represents acomplex of cryptic sibling species. Previous studies suggested the existence of four main clades in LatinAmerica representing different species with distinct geographical ranges: (1) Brazil (except RoraimaState) – Species A = Lu. longipalpis sensu stricto, (2) Laran – Species B = Lu. pseudolongipalpis(Venezuela), (3) cis-Andean – Species C (Colombia and northwestern Venezuela, and Roraima State inBrazil) and (4) trans-Andean – Species D (Central America). In addition, cross-mating experiments,analysis of copulation songs, pheromones, microsatellites and lovesong genes, suggested that the Brazilianpopulations of this vector represent different species that can be divided into two main groups accordingto the type of song (Burst vs. Pulse) males produce during copulation. Nevertheless, no diagnosticdifferences have been observed between these two groups with most molecular markers used to date. Weinitially analyzed the molecular divergence in a fragment of the paralytic gene, a locus involved in thecontrol of courtship songs in Drosophila, among a number of Lu. longipalpis populations from Brazilproducing Burst and Pulse-type songs. We also compared Lu. longipalpis with a very closely relatedspecies, Lutzomyia cruzi, which produces Burst-type song. The results indicated a higher number of fixeddifferences between Lu. cruzi and the Pulse-type populations of Lu. longipalpis than with those producingBurst-type songs. The data confirmed that the different sibling species of the Lu. longipalpis complex inBrazil can be divided into two main groups, one representing a single species and a second, a moreheterogeneous group, that probably represents a number of incipient species. We also performed amacrogeographic analysis of the divergence and gene flow in the paralytic gene among Lu. longipalpispopulations from South and Central America...

Genetic differentiation in natural populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae) with different phenotypic spot patterns on tergites in males

Entomological surveys in the state of Maranhão have recorded morphologically distinct populations of Lutzomyia longipalpis (Lutz & Neiva). Some populations have one pair of spots (1S) on the fourth tergite, while others have two pairs (2S) on the third and fourth tergites of males. In the present study we investigated the degree of genetic polymorphism among four populations in the municipalities of Caxias, Codó and Raposa, in the state of Maranhão, Brazil, by using RAPD (Random Amplified Polymorphic DNA) markers. A total of 35 loci were identified, of which 30 were polymorphic. The highest polymorphism was observed with primer OPA 4, which produced 11 different profiles. Genetic diversity was assessed using grouping methods that produced a dendrogram in which the genotypes could be clearly separated into two main clades according to the number of spots on the male abdominal tergites. One cluster contained the populations from Caxias and Codó, and the other was formed by the populations from Raposa and Codó. The results of our RAPD analysis showed a clear separation between the populations with one and two pairs of spots. The epidemiologic significance of this genetic differentiation should be investigated in future studies.

Infecção natural de Lutzomyia (Lutzomyia) longipalpis (Diptera: Psychodidae) por Leishmania infantum chagasi em flebotomíneos capturados no município de Janaúba, Estado de Minas Gerais, Brasil / Natural infection with Leishmania infantum chagasi in Lutzomyia (Lutzomyia) longipalpis (Diptera: Psychodidae) sandflies captured in the municipality of Janaúba, State of Minas Gerais, Brazil

INTRODUÇÃO: A leishmaniose visceral tem sido notificada em quase todos os estados do Brasil, e principalmente no norte de Minas Gerais, onde a doença é endêmica. Este estudo visou detectar a infecção natural de Lutzomyia longipalpis e identificar através da técnica de PCR/RFLP a espécie de Leishmania encontrada nos flebotomíneos do município de Janaúba. MÉTODOS: Utilizando-se armadilhas luminosas, foram capturadas 1.550 fêmeas de L. longipalpis, que agrupadas em pool de 10 exemplares foram submetidas à extração e amplificação de DNA, através das técnicas de PCR genérico e cacofonia. RESULTADOS: Dos 155 pools, seis apresentaram-se positivos para Leishmania sp., sendo a taxa de infecção do município de 3,9 por cento. Através da PCR/RFLP determinou-se que o padrão de digestão das amostras positivas foi semelhante ao da cepa referência Leishmania chagasi (MHOM/BR/74/PP75). CONCLUSÕES: A detecção de infecção natural associada a estudos sobre a epidemiologia da LV sugere que L. longipalpis esteja envolvida na transmissão de L. infantum chagasi em Janaúba, principalmente nas áreas de intensa transmissão de LV.

INTRODUCTION: Visceral leishmaniasis has been notified in nearly all states of Brazil, and particularly in the north of Minas Gerais, where the disease is endemic. The aim of this study was to detect natural infection of Lutzomyia longipalpis and, through the PCR/RFLP technique, identify Leishmania species found in sandflies in the municipality of Janaúba. METHODS: Using light traps, 1,550 females of L. longipalpis were caught and grouped into pools of 10 specimens to be subjected to DNA extraction and amplification, by means of generic PCR and cacophony. RESULTS: Out of the 155 pools, six were positive for Leishmania sp., and thus the infection rate in the municipality was 3.9 percent. Through PCR/RFLP, the digestion pattern among the positive samples was found to be similar to that of the reference strain of Leishmania chagasi (MHOM/BR/74/PP75). CONCLUSIONS: The detection of natural infection associated with studies on the epidemiology of visceral leishmaniasis suggests that L. longipalpis is involved in transmission of L. infantum chagasi in Janaúba, particularly in areas of intense transmission of visceral leishmaniasis.

Aislamiento reproductivo asimétrico entre Lutzomyia pseudolongipalpis y Lutzomyia longipalpis (especie C2), vectores neotropicales de leishmaniasis visceral (Diptera: Pshychodidae)

Asymmetric reproductive isolation between Lutzomyia pseudolongipalpis and Lutzomyia longipalpis (species C2), Neotropical vectors of visceral leishmaniasis (Diptera: Pshychodidae). Lutzomyia pseudolongipalpis and Lutzomyia longipalpis (species C2, L. sp n.) are two endemic species of Phlebotominae sand fly vectors from Venezuela. The two insects are sympatric and monophyletic but have deeply diverging, morphological differences. They belong to the L. longipalpis complex. A study of their reproductive isolation is necessary to understand the process of speciation and maintenance of the two sister species as two discrete taxonomic and biological entities. Cross-mating tests were conducted (homo and hetero-specific) and monitored under two criteria: biological (presence of copulation and offspring) and genetic (using two isozymic markers diagnostic for the L. longipalpis complex; enzyme diagnosis AK and HK). Results indicate reproductive isolation, with an asymmetrical genetic exchange in a direction of hybridization between the two species under experimental conditions, and production of a low number of heterozygotes. These findings support the existence of negative selection on hybrids, and explain the absence of hybrids under natural conditions, in the sympatric locality. Rev. Biol. Trop. 57 (1-2): 23-31. Epub 2009 June 30.

Lutzomyia pseudolongipalpis y Lutzomyia longipalpis (especie C2, L. n. sp) son dos especies de flebotominos vectores endémicos de Venezuela, simpátricas, monofiléticas, con profundas divergencias, morfológicamente diferentes y pertenecientes al complejo de especies L. longipalpis. El estudio de su aislamiento reproductivo es clave para entender el proceso de especiación y el mantenimiento de estas dos especies hermanas como entidades taxonómicas y biológicas discretas. Por tal motivo, se realizaron ensayos de entrecruzamiento homo y heteroespecíficos, los cuales fueron monitoreados por dos criterios: biológico (presencia de cópula y progenie) y genético (utilizando dos marcadores isoenzimáticos diagnósticos para el complejo L. longipalpis, las enzimas AK y HK). Los resultados indican aislamiento reproductivo, con un intercambio genético asimétrico hacia la hibridización en condiciones experimentales, y producción de un bajo número de heterocigotos, lo que apoya la existencia de selección negativa sobre los híbridos y explica su ausencia en condiciones naturales en la localidad simpátrica.

Characterization and speculations on the urbanization of visceral leishmaniasis in Brazil / Caracterização e especulações acerca da urbanização da leishmaniose visceral no Brasil

The available hypotheses or explanations for the urbanization of American visceral leishmaniasis are insufficient. An alternative hypothesis is that changes in the ecology and biology of the vector, Lutzomyia longipalpis, might explain all the new urban epidemiological features of the disease. To tackle the knowledge gaps in this process, certain key research areas need to be prioritized: the role of dogs in expanding transmission in cities, community trials to evaluate new insecticides, and research on the ecological and molecular determinants of Leishmania chagasi transmission. Investment of public funds should focus on the development of a human vaccine, since such a vaccine now appears to be within reach. Even small effects from a vaccine could substantially reduce the impact of the disease, which in the last quarter century has challenged and defeated both the scientific community and the public health field around the world.

As hipóteses ou explicações apresentadas até o momento para o processo de urbanização da leishmaniose visceral americana são insatisfatórias. Uma hipótese alternativa é a de que mudanças na ecologia e biologia do vetor, Lutzomyia longipalpis, poderiam explicar as feições epidemiológicas urbanas da doença. De forma a suprir lacunas no conhecimento sobre esse processo de urbanização, destacam-se algumas linhas de pesquisa prioritárias: a investigação do papel de cães na amplificação da transmissão da doença nas cidades, ensaios de campo com novos inseticidas, investigação dos determinantes ecológicos ou moleculares que participam da transmissão de Leishmania chagasi. Esforços com investimentos públicos devem ser feitos para o desenvolvimento de vacinas para humanos, pois uma vacina não parece estar tão distante. Resultados vacinais mesmo modestos podem reduzir substancialmente o impacto dessa doença que, no último quarto de século, desafiou e venceu a ciência e a saúde pública mundo afora.

Estructura genética en cinco especies de flebótomos (Lutzomyia spp.) de la serie townsendi, grupo verrucarum, en Colombia (Diptera: Prychodidae)

Genetic structure in five Phlebotominae (Lutzomyia spp.), townsendi series, verrucarum group, in Colombia (Diptera: Prychodidae). Sixteen isoenzyme patterns were analyzed for five Colombian Lutzomyia species. The average unbiased expected heterozygosity levels ranged from 0.098 (Lu. youngi) to 0.215 (Lu. torvida). The five species samples, taken all the isoenzymes employed, were significantly deviated from the Hardy-Weinberg equilibrium by homozygous excess with classical as well as Markov chain exact tests. Possible causes: (1) Wahlund effect within populations due to subdivision and/or sampling. Endogamy could be discarded because these loci were affected by highly different levels of homozygous excess. (2) Null alleles could be not discarded, at least for some isoenzymes. The hierarchical Wright´s F analysis showed high and significant values for each parameter. The average F IT value was 0.655 with a conspicous homozygous excess at a global level (all species taken together); the average F IS value was significantly positive (0.515) as well, with homozygous excess within each species. The genetic heterogeneity between the fives species was noteworthy (F ST = 0.288), indicating clear genetic differentiation. The more related species pairs were Lu. longiflocosa-Lu. torvida (0.959) and Lu torvida-Lu. spinicrassa (0.960); while Lu. torvida-Lu. youngi (0.805) and Lu. quasitownsendi-Lu. youngi (0.796) were the most divergent (Nei´s genetic identity matrix). UPGMA and Wagner algorithms showed that the most divergent species was Lu. youngi, whereas the most related were Lu. longiflocosa-Lu. torvida and Lu torvida-Lu. spinicrassa. A spatial autocorrelation analysis (Moran´s I index) revealed a very weak, or inexistent spatial structure, which means that the speciation events between these species were independent from the geographic distances from where they currently live. Rev. Biol. Trop. 56 (4): 1717-1739. Epub 2008 December 12.

Se analizaron 16 sistemas isoenzimáticos para cinco especies colombianas del género Lutzomyia. Los niveles de heterocigosis media esperada insesgada oscilaron entre 0.098 (Lu. youngi) y 0.215 (Lu. torvida). Las cinco muestras estudiadas de forma global, para todos los marcadores analizados, presentaron desviación respecto al equilibrio Hardy-Weinberg por un exceso de homocigotos, tanto al utilizar algunas pruebas clásicas como tests exactos con cadenas de Markov. Este hecho puede estar favorecido por diversas causas: (1) la más probable es la existencia de efecto Wahlund en el seno de cada población debido a subdivisión y/o a la técnica de muestreo empleada. La endogamia puede descartarse ya que no todos los loci están afectados por el mismo tipo de exceso de homocigotos. (2) Sin embargo, no se puede descartar la existencia de alelos nulos, al menos, para algunos de los marcadores isoenzimáticos utilizados. El análisis jerarquizado con las F de Wright mostró valores elevados y significativos para cada uno de los estadísticos. El estadístico promedio F IT mostró un valor de 0.655 existiendo un conspicuo exceso de homocigotos a nivel total de todas las especies, el estadístico promedio F IS fue altamente positivo (0.515) mostrando exceso de homocigotos a nivel individual en cada una de las especies estudiadas. La heterogeneidad genética entre las cinco especies fue notable (F ST = 0.288). Esto muestra que esas especies están bien diferenciadas a nivel isoenzimático y que en el interior de cada especie también hay una subdivisión genética. La matriz de identidades genéticas de Nei muestra que las especies más relacionadas fueron Lu. longiflocosa-Lu. torvida (0.959) y Lu torvida-Lu. spinicrassa (0.960) mientras que las genéticamente más distantes fueron Lu. torvida-Lu. youngi (0.805) y Lu. quasitownsendi-Lu. youngi (0.796). Con los algoritmos UPGMA y Wagner, se observó que la especie más divergente fue Lu. youngi, mientras que las relaciones más conspicuas se observaron entre Lu. longiflocosa-Lu. torvida y Lu torvida-Lu. spinicrassa. Adicionalmente, con un análisis de autocorrelación espacial (índice de Moran) la mayoría de los alelos utilizados presentaron una estructura espacial muy débil o inexistente, lo que significa que los eventos de especiación entre las especies estudiadas se dieron en forma independiente de las distancias geográficas existentes actualmente entre ellas.

Genetic divergence between two sympatric species of the Lutzomyia longipalpis complex in the paralytic gene, a locus associated with insecticide resistance and lovesong production

The sandfly Lutzomyia longipalpis s.l. is the main vector of American Visceral Leishmaniasis. L. longipalpis s.l. is a species complex but until recently the existence of cryptic sibling species among Brazilian populations was a controversial issue. A fragment of paralytic (para), a voltage dependent sodium channel gene associated with insecticide resistance and courtship song production in Drosophila, was isolated and used as a molecular marker to study the divergence between two sympatric siblings of the L. longipalpis complex from Sobral, Brazil. The results revealed para as the first single locus DNA marker presenting fixed differences between the two species in this locality. In addition, two low frequency amino-acid changes in an otherwise very conserved region of the channel were observed, raising the possibility that it might be associated with incipient resistance in this vector. To the best of our knowledge, the present study represents the first population genetics analysis of insecticide resistance genes in this important leishmaniasis vector.

Descripción del ARN de transferencia mitocondrial para Serina (UCN) de Lutzomyia columbiana (Diptera, Psychodidae) / Description of the mitochondrial serine transfer RNA (UCN) of Lutzomyia columbiana (Diptera, Psychodidae)

Lutzomyia columbiana es un flebotomíneo considerado como vector sospechoso de Leishmania mexicana y Leishmania braziliensis en Colombia. Este insecto pertenece al grupo verrucarum, que incluye algunos taxones isomórficos, lo que ha estimulado la búsqueda de marcadores moleculares que permitan, además de diferenciar las especies, estudiar sus relaciones de parentesco. En este artículo se describe por primera vez la estructura putativa del ARN de transferencia mitocondrial para serina que reconoce el codón UCN (ARNtSer) de Lu. columbiana. El ADN genómico fue extraído, amplificado y secuenciado a partir de seis especímenes colectados con cebo humano. La estructura secundaria del ARNtSer fue inferida con el programa tRNAscan-SE 1.21. El gen ARNts consistió de 67 pares de bases (pb), encontrándose un solo haplotipo en los seis individuos secuenciados. El ARNtSer de Lu. columbiana mostró 7 apareamientos intracatenarios en el brazo aceptor del aminoácido, 3 en el brazo dihidrouridina (DHU), 5 en el brazo del anticodón y 5 en el brazo ribotimidina-pseudouridina-citosina (T›C). El tamaño de las lupas correspondió a 5 nucleótidos en la DHU, 7 en la anticodón, 4 en la variable y 7 en la T›C. Lu. columbiana se distingue del resto de especies de Lutzomyia y Phlebotomus secuenciadas a la fecha por la presencia de una guanina en la posición nucleotídica 64, que produce un apareamiento no canónico tipo uracilo-guanina en el brazo aceptor. Se necesitan más estudios para confirmar la utilidad del ARNtSer como marcador molecular para la discriminación de especies de flebotomíneos.

The sand fly Lutzomyia columbiana is considered a suspected vector of Leishmania mexicana and Leishmania braziliensis in Colombia. Lu. columbiana belongs to the Lutzomyia verrucarum species group, which included some sibling species. This has motivated the search for molecular markers to distinguish these taxa. In this paper, we described for the first time the putative secondary structure of the mitochondrial serine transfer RNA that recognizes the codon UCN of Lu. columbiana (tRNA Ser). DNA was extracted, amplified and sequenced from six individuals collected in human biting activity. The secondary structure of the tRNA Ser was inferred using the program tRNAscan-SE 1.21. The tRNA Ser gene length was 67 pair of bases (pb), and a single haplotype was detected among the six specimens sequenced. In the inferred secondary structure of the tRNA Ser of Lu. columbiana, the acceptor arm consisted of 7 bp, the dihydrouridine (DHU) arm of 3 pb, the anticodon arm of 5 pb, and the ribothymidine-pseudouridine-cytosine (T›C) arm of 5 pb. Similarity, the estimated size of the loops was 5 nucleotides in the DHU, 7 in the anticodon, 4 in the variable, and 7 in the T›C. Lu. columbiana differs from other Lutzomyia and Phlebotomus species sequenced to date by the presence of guanine in the nucleotide position 64, which induce a non-canonical base pair conformation type uracil-guanine in the acceptor arm. More studies are necessary to confirm the usefulness of the tRNA Ser as a suitable molecular tool for sand fly species identification.