Fixed differences in the paralytic gene define two lineages within the Lutzomyia longipalpis complex producing different types of courtship songs.

The sand fly Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae), the most important vector of American visceral leishmaniasis, is widely distributed in Latin America. There is currently a consensus that it represents a species complex, however, the number and distribution of the different siblings is still uncertain. Previous analyses have indicated that Brazilian populations of this vector can be divided into two main groups according to the type of courtship song (Burst vs. Pulse) males produce during copulation. Nevertheless, no diagnostic differences have been observed between these two groups with most molecular markers used to date. We analyzed the molecular divergence in a fragment of the paralytic (para) gene, a locus involved in the control of courtship songs in Drosophila, among a number of Lu. longipalpis populations from Brazil producing Burst and Pulse-type songs. Our results revealed a very high level of divergence and fixed differences between populations producing the two types of songs. We also compared Lu. longipalpis with a very closely related species, Lutzomyia cruzi, which produces Burst-type songs. The results indicated a higher number of fixed differences between Lu. cruzi and the Pulse-type populations of Lu. longipalpis than with those producing Burst-type songs. The data confirmed our previous assumptions that the presence of different sibling species of the Lu. longipalpis complex in Brazil can be divided into two main groups, one representing a single species and a second more heterogeneous group that probably represents a number of incipient species. We hypothesize that para might be one of the genes directly involved in the control of the courtship song differences between these two groups or that it is linked to other loci associated with reproductive isolation of the Brazilian species.

SYBR Green-based real-time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniases.

BACKGROUND: Leishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures. Accurate diagnosis, which can distinguish between Leishmania isolates, is essential for conducting appropriate prognosis, therapy and epidemiology. Molecular methods are currently being employed to detect Leishmania infection and categorize the parasites up to genus, complex or species level. Real-time PCR offers several advantages over traditional PCR, including faster processing time, higher sensitivity and decreased contamination risk. RESULTS: A SYBR Green real-time PCR targeting the conserved region of kinetoplast DNA minicircles was able to differentiate between Leishmania subgenera. A panel of reference strains representing subgenera Leishmania and Viannia was evaluated by the derivative dissociation curve analyses of the amplified fragment. Distinct values for the average melting temperature were observed, being 78.95 °C ± 0.01 and 77.36 °C ± 0.02 for Leishmania and Viannia, respectively (p < 0.05). Using the Neighbor-Joining method and Kimura 2-parameters, the alignment of 12 sequences from the amplified conserved minicircles segment grouped together L. (V.) braziliensis and L. (V.) shawii with a bootstrap value of 100%; while for L. (L.) infantum and L. (L.) amazonensis, two groups were formed with bootstrap values of 100% and 62%, respectively. The lower dissociation temperature observed for the subgenus Viannia amplicons could be due to a lower proportion of guanine/cytosine sites (43.6%) when compared to species from subgenus Leishmania (average of 48.4%). The method was validated with 30 clinical specimens from visceral or cutaneous leishmaniases patients living in Brazil and also with DNA samples from naturally infected Lutzomyia spp. captured in two Brazilian localities. CONCLUSIONS: For all tested samples, a characteristic amplicon melting profile was evidenced for each Leishmania subgenus, corroborating the data from reference strains. Therefore, the analysis of thermal dissociation curves targeting the conserved kinetoplast DNA minicircles region is able to provide a rapid and reliable method to identify the main etiologic agents of cutaneous and visceral leishmaniases in endemic regions of Brazil.

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...

Voltage-gated sodium channel polymorphism and metabolic resistance in pyrethroid-resistant Aedes aegypti from Brazil.

The nature of pyrethroid resistance in Aedes aegypti Brazilian populations was investigated. Quantification of enzymes related to metabolic resistance in two distinct populations, located in the Northeast and Southeast regions, revealed increases in Glutathione-S-transferase (GST) and Esterase levels. Additionally, polymorphism was found in the IIS6 region of Ae. aegypti voltage-gated sodium channel (AaNa(V)), the pyrethroid target site. Sequences were classified in two haplotype groups, A and B, according to the size of the intron in that region. Rockefeller, a susceptible control lineage, contains only B sequences. In field populations, some A sequences present a substitution in the 1011 site (Ile/Met). When resistant and susceptible individuals were compared, the frequency of both A (with the Met mutation) and B sequences were slightly increased in resistant specimens. The involvement of the AaNa(V) polymorphism in pyrethroid resistance and the metabolic mechanisms that lead to potential cross-resistance between organophosphate and pyrethroids are discussed.

Isolamento e genética de populações de genes associados à resistência a inseticidas em Lutzomyia longipalpis (Lutz & Neiva,1912) (Diptera: Psychodidae: Phlebotominae), vetor da Leishmaniose Visceral Americana / Isolation and genetics of populations of genes associates to the resistance the insecticides in Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae), vector of the American Visceral Leishmaniasis

Flebotomíneos são os insetos vetores das leishmanioses e Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) o principal transmissor da Leishmania infantum chagasi, agente etiológico da Leishmaniose Visceral Americana (LVA). Devido a sua importância como vetor, diversos estudos foram conduzidos de forma a verificar o real status taxonômico de L. longipalpis. A existência de um complexo de espécies crípticas entre populações brasileiras ainda é conflituosa. No presente trabalho, foram isolados fragmentos de três genes associados à resistência a inseticidas, os genes paralytic (para), Acetylcholinesterase (Ace) e Resistant to dielrin (Rdl). Foi escolhida uma região do gene para onde algumas mutações para resistência a piretróides e DDT ocorrem em outras espécies de insetos, para ser utilizada como marcador para estudar populações brasileiras de L. longipalpis e para investigar a ocorrência de mutações potencialmente associadas à resistência a inseticidas neste vetor. Amostras das localidades de Sobral (CE), Pancas (ES), Lapinha (MG), Jacobina (BA), Estrela de Alagoas (AL) e Nísia Floresta, Grande Natal (RN) foram analisadas. Os resultados obtidos com o gene para, corroboram resultados anteriores mostrando que em Sobral, machos com os fenótipos de uma (1S) e duas (2S) pintas abdominais pertencem a duas espécies crípticas vivendo em simpatria. A ocorrência de diferenças fixas no fragmento estudado entre os dois tipos de machos, torna para um marcador molecular muito útil para identificar fêmeas dessas duas espécies em estudos futuros. Os dados apresentados também sugerem que Pancas, Sobral 2S e Natal pertencem à mesma espécie, enquanto Sobral 1S, Lapinha, Jacobina e Estrela 1S, provavelmente representariam quatro outras espécies no Brasil. Os resultados obtidos estão de acordo com estudos anteriores utilizando outros marcadores moleculares, experimentos de cruzamentos, feromônio e análise de som de cópula, e sugerem fortemente a existência de um complexo ...