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Background: Entomological surveillance of sand fly vectors was carried out to support leishmaniasis prevention and control measures in areas affected by the São Francisco River Transposition Project., Methods: Sand flies were collected monthly between May 2011 and December 2014 in seven municipalities: Missão Velha, Brejo Santo, Lavras da Mangabeira, Iguatu, Mauriti, Jaguaribe and Jaguaretama, in dwellings, peridomicile and forest areas for three consecutive days, for a period of 12 h each day (18:00 to 06:00 h). Differences in species composition between sites were tested with Shannon's diversity index, the similarity between habitats was estimated by the Sørensen's qualitative similarity index and, for the most abundant species in each municipality, a standardized index of species abundance was applied. The influence of climatic factors on sand fly population densities was analyzed using Spearman's correlation coefficients., Results: A total of 214,213 sand fly specimens belonging to 18 species were captured. The most abundant species in all municipalities was Lutzomyia longipalpis (Lutz & Neiva, 1912). The municipalities of Mauriti and Missão Velha stand out in terms of high species richness, with the latter exhibiting the greatest diversity. The number of sand flies in the Iguatu, Jaguaribe and Jaguaretama municipality was higher during the rainy months, whereas the populations declined in the drier months; the sand fly population density in other municipalities (Missão Velha, Brejo Santo, Lavras de Mangabeira and Mauriti) showed negative correlation with rainfall., Conclusions: This study confirms the presence of several Leishmania spp. vectors in the seven municipalities affected by the São Francisco River Transposition Project, with Lu. longipalpis being the most abundant species at all study sites. Vector populations in these municipalities should be monitored, ultimately to assess the associations between environmental changes and sand fly population dynamics and leishmaniasis transmission risk.

The immunization of vertebrate hosts with vector components may be an alternative for the control of diseases transmitted by insects. In the present study we evaluated the effects of anti-sandfly antibodies on some of the biological parameters of female Lutzomyia longipalpis, a vector of visceral leishmaniasis. Rabbits were immunized with extracts of gut from blood-fed (GB) or sugar-fed (GS) females, carcass of sugar-fed (CS) or blood-fed (CB) females, and with repeated sandfly bites (BITE). Immune sera showed increased antibody titers compared to pre-immunized animals, and specific bands were detected by Western Blot. An analysis of biological parameters revealed a decline in fecundity in the group of females fed on rabbits immunized with GB and BITE. Longevity and mortality were studied in females with oviposition (parous) and without oviposition (nulliparous). Nulliparous females that fed on rabbits immunized with bites died in the highest percentage. A mortality analysis after egg laying revealed a peak on the fifth day in all the groups, but females fed on rabbit subjected to repeated bites showed a shift towards the third day.

The genetic relationships among male Lutzomyia (Nyssomyia) intermedia (Lutz & Neiva) (Diptera: Psychodidae) from three populations from the same endemic area of American cutaneous leishmaniasis (ACL) in the state of Rio de Janeiro, Brazil, were compared. The sandflies were collected in three ecologically different habitats: domestic, extra-domestic and sylvatic over a total range of 800 m. Three molecular markers were employed to assess population variation. Based on MLEE markers, it could not be concluded that the three populations do not belong to the same gene pool (F(st) = 0.005). No within-population departure from Hardy-Weinberg equilibrium was detected (P < 0.05) and they presented the same level of gene variation. The number of migrants (Nm) indicated that at least 50 individuals per generation migrated between the three habitats. RAPD-PCR markers revealed that, except for the primer five, all were polymorphic. Phenetic analysis of the genotypes showed the presence of two principal clusters corresponding to: (1) domestic plus extra-domestic and (2) sylvatic. Unique genotypes were observed in each population. The sylvatic population was the most polymorphic, showing the largest number of genotypes and low level of similarity between them. Three mtDNA gene markers were studied by SSCP analysis. The most frequent haplotype for each marker ranged in frequency from 60 to 87% and individuals with unique haplotypes varied from 1 to 5%. Interestingly, the SSCP analysis showed a low level of polymorphism within populations. The disagreement between the different molecular markers observed and the hypothesis that L. intermedia could be participating in the transmission cycle of Leishmania (Viannia) braziliensis in environments ranging from the interior of human dwellings to the forest, are discussed.

During two consecutive years, studies on the sand fly fauna in Poço das Antas and Fazenda Bom Retiro, two Atlantic Rain Forest Reserves from the State of Rio de Janeiro, were performed using Shannon traps, CDC light traps and human bait collections. Eleven species were identified; Lutzomyia longipalpis, L. migonei, L. edwardsi, L. intermedia, L. whitmani, L. fischeri, L. shannoni, L. ayrozai, L. hirsuta, L. monticola and L. misionensis (first occurrence in the State of Rio de Janeiro). L. intermedia and L. whitmani were the predominant anthropophilic species around houses, while L. hirsuta predominated in the forest.

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

Studies were performed on five Brazilian populations of Lutzomyia longipalpis: Salvaterra (PA), São José do Ribamar (MA), Canindé (CE), Natal (RN) and Gruta da Lapinha, Lagoa Santa (MG). No morphological differences were observed that could distinguish between these populations. Homogeneity tests showed that the allopatric populations display a certain heterogeneity and that the sympatric populations, with different patterns of spots, are homogeneous. The Student-Newman-Keuls test, represented by Euler-Venn diagrams, showed a disjunction between the populations from the north/northeast and the one from Gruta da Lapinha. Genetic distances between the four populations (excluding the Canindé population) were within the range of intrapopulational differences. The Gruta da Lapinha population displayed a heterozygotic deficiency that could be a consequence of high levels of inbreeding due to cryptic habits of living in a small cave. These results do not favor the hypothesis of a L. longipalpis species complex in Brazil, and the species should be considered high polymorphic.

Eleven populations of Lutzomyia longipalpis (Lutz & Neiva), the sand fly vector of Leishmania chagasi, from different areas of Brazil were analyzed for genetic variation at 16 enzyme loci. In this region, the prevalence of visceral leishmaniasis (VL) caused by L. chagasi is spotty and reproductive isolation among populations of Lu. longipalpis has been reported. It is thought that morphologically similar cryptic species with varying vectorial capacity may be responsible for the discontinuous distribution of VL. The aim was to study the genetic structure of populations within this region and to identify demes that may represent sibling species. Genotypic frequencies within populations were in close compliance to Hardy-Weinberg expectations, suggesting there are no sympatric species among these 11 populations. Levels of genetic distance between pairs of populations were very low (< 0.03), consistent with local populations within a single sand fly species. When genotypic frequency data for all populations were pooled, 9 of the 13 polymorphic loci deviated from Hardy-Weinberg expectations, indicating some degree of genetic substructuring. Estimates of effective migration rates (N(e)m) among all populations were low, 2.73, suggesting that gene flow is restricted among populations, which is probably the reason for the observed genetic substructuring.

Phylogenetic analysis of all 31 described mitochondrial (cytochrome b) haplotypes of Lutzomyia whitmani demonstrated that new material from the State of Rondônia, in southwest Amazônia, forms a clade within a lineage found only in the rain-forest regions of Brazil. This rain-forest lineage also contains two other clades of haplotypes, one from eastern Amazônia and one from the Atlantic forest zone of northeast Brazil (including the type locality of the species in Ilhéus, State of Bahia). These findings do not favour recognizing two allopatric cryptic species of L. whitmani, one associated with the silvatic transmission of Leishmania shawi in southeast Amazônia and the other with the peridomestic transmission of Le. braziliensis in northeast Brazil. Instead, they suggest that there is (or has been in the recent past) a continuum of inter-breeding populations of L. whitmani in the rain-forest regions of Brazil.

The phlebotomine sand fly Lutzomyia longipalpis is the insect vector of visceral leishmaniasis, a protozoan disease of increasing incidence and distribution in Central and South America. Electrophoretic allele frequencies of 15 enzyme loci were compared among the L. longipalpis populations selected across its distribution range in Brazil. The mean heterozygosity of two colonized geographic strains (one each from Colombia and Brazil) were 6% and 13% respectively, with 1.6-1.9 alleles detected per locus. In contrast, among the seven widely separated field populations, the mean heterozygosity ranged from 11% to 16% with 2.1-2.9 alleles per locus. No locus was recovered that was diagnostic for any of the field populations. Allelic frequency differences among five field strains from the Amazon basin and eastern coastal Brazil were very low, with Nei's genetic distances of less than 0.01 separating them. The two inland and southerly samples from Minas Gerais (Lapinha) and Bahia (Jacobina) states were more distinctive with genetic distances of 0.024-0.038 and 0.038-0.059, respectively, when compared with the five other samples. These differences were the consequence of several high frequency alleles (glycerol-3-phosphate dehydrogenase [Gpd1.69] and phosphoglucomutase [Pgm1.69]) relatively uncommon in other strains. The low genetic distances, absence of diagnostic loci, and the distribution of genes in geographic space indicate L. longipalpis of Brazil to be a single, but genetically heterogeneous, polymorphic species.

The phlebotomine sand fly, Lutzomyia longipalpis, is the vector of visceral leishmaniasis in the New World. Variability in its tergal spot morphology has led to conflicting interpretations of the species status of the various forms. An L. longipalpis field population from eastern Brazil was found with three co-occurring morphological variations--1-spot, 2-spot, and an intermediate form. Genetic profiles were established for each form. Fifteen isoenzyme loci provided the data matrix for comparison of genetic variation among the forms. Spot patterns and isoenzyme frequencies fit Hardy-Weinberg expectations, and no significant differences in isoenzyme frequencies were associated with morphological phenotype. The spot phenotype appears to be a polymorphic character not related to genetic isolation or differentiation at the species level.

The genetic qualities of laboratory colonies of phlebotomine sand flies have not been compared with field specimens despite 1) probable genetic shifts due to the colonization process and 2) the problems associated with the extrapolation of experimental data derived from colonized organisms to field populations. The present study compared the genetic profiles of five laboratory colonies of geographic strains of the New World sand fly Lutzomyia longipalpis, and contrasted them with field populations. The profiles were based on the variability exhibited with polyacrylamide gels at 14 enzyme loci. A general pattern of a loss of infrequent alleles and decreased heterozygosity emerged as an apparent consequence of colonization. The average number of alleles per locus ranged from 1.2 to 1.6, and the average heterozygosity ranged from 4% to 11%. The field collection from Lapinha Caves (near Belo Horizonte, Brazil) averaged 2.1 alleles with a heterozygosity of 16%. In contrast, the LAPINHA laboratory colony established from that site 24 years earlier showed very low values (1.2 alleles/locus and 4% heterozygosity) and fixation for alleles not present or rare in the field collection from the same site. The genetic differences between the other Brazilian colonies and the Lapinha Caves field samples were due to presence of both different alleles and highly diverged allelic frequencies. Biological inferences based on colonized sand flies must be tempered by recognizing that the colony may represent a highly skewed genetic subsample of the L. longipalpis field genome.

Phylogenetic analysis of morphometric and biological characters indicated that there are two distinct forms of Lutzomyia whitmani in Brazil: one is present both north and south of the River Amazonas in the State of Pará while the other occurs in northeast Brazil, in the State of Ceará, and further south, including the type locality in State of Bahia. The Amazonian form is reportedly neither strongly anthropophilic nor synanthropic, and it is the vector of Leishmania shawi; whereas the southern form is often collected peridomestically, while biting man, and has been found infected with Le.(V.) braziliensis. The ratio of the length of the genital filaments to that the genital pump was found to be consistently smaller in males of the Amazonian populations. A middle repetitive DNA element was isolated by differentially screening a genomic library made using Amazonian material, and the sequence was diagnostic for this form of Lu. whitmani (being absent or occurring in low copy number in the southern form). The total evidence suggests there are at least two, geographically-isolated forms of Lu. whitmani, which may represent different cryptic species.

The municipality of Ilhéus, State of Bahia, has a focus of cutaneous leishmaniasis where entomological studies were carried out to determine the sand fly species and their habits. Lutzomyia migonei, L. sallesi, L. tupynambai, L. schreiberi, L. intermedia, L. whitmani, L. yuilli yuilli, L. fischeri, L. pessoai, L. shannoni and L. misionensis were identified. Lutzomyia whitmani was the predominant species. Specimens were collected indoors, at peridomestic sites, in the cocoa plantations and in other types of collections. Females fed readily on humans and were attracted to domestic animals. Our evidence suggests that L. whitmani is a probable vector.

Schneider's Drosophila medium, a complex amino acid rich medium was tested alone and with seven different sugars for some aspects of the biology of Lutzomyia longipalpis. Statistically significant results were obtained when sucrose was used alone, indicating that among the sugars tested, this is still the most suitable and practical one for the maintenance of L. longipalpis colonies. However, the addition of Schneider's medium to a pool of different sugars, was suggested to be related with the acceptance of the first and second blood meals and to longevity, these being, obviously, quite relevant aspects when transmission experiments are contemplated.

The development of Colombian Leishmania species of the subgenus Viannia in Lutzomyia intermedia was similar to that observed with Brazilian Le. (V.) braziliensis: colonization of the pylorus by paramastigotes; promastigotes in the midgut and massive infection of stomodeal valve. Difference was observed in the number of paramastigotes colonizing the pylorus, which was smaller in Colombian Leishmania species than Brazilian Le. braziliensis.

The development of Leishmania (Viannia) braziliensis in experimentally infected Lutzomyia intermedia, showed colonization of the hindgut from 48 h after the infective blood-meal, and the migration of flagellates to the foregut, with a massive infection of the cardia at the 5th day post infection. Up to 10 days following the infective blood-meal, very few parasites were seen in the pharynx and cibarium. The role of L. intermedia as a vector of cutaneous leishmaniasis is discussed according to the established criteria.

In order to be comparable, and efficient, the descriptions need to be as complete as possible and realized according to the same parameters. In this respect, we present the standard description of Phlebotomine sandflies elaborated by an American network of entomologists in a collaborative program dedicated to the creation of a Computer Aided Identification Program for Phlebotomine Sandflies of South and Central America (CIPA programme).

In a study on putative vectors of cutaneous leishmaniasis in Baturité, Ceará State, Brazil, six sandfly species were identified: Lutzomyia longipalpis, L. wellcomei, L. peresi, L. whitmani, L. shannoni and L. migonei. In general L. whitmani and L. migonei were the predominant species. They were collected in periodomiciliary areas and were attracted by man and equines. L. whitmani was the most anthropophilic species. Studies using animals as bait showed that blood-feeding occurs throughout the night. Previously L. whitmani had been found infected with a Leishmania species of the braziliensis complex and L. migonei was infected with peripylarian flagellates. In combination these findings suggest that they may be responsible for periodomestic transmission of cutaneous leishmaniasis.

In a study on vectors of cutaneous leishmaniasis in Mesquita, Rio de Janeiro State, Brazil, eleven species of sandflies were found: Lutzomyia longipalpis, L. fischeri, L. quinquefer, L. intermedia, L. whitmani, L. shannoni, L. cortelezzii, L. edwardsi, L. migonei, L. lanei and L. firmatoi. Lutzomyia intermedia was the predominant species. It was collected indoors but in much higher numbers outdoors, in the close vicinity of houses. In comparative captures it was found to feed more frequently on equines, and less frequently on man and dogs. Studies, using man and animals as baits, showed that blood-feeding started in the evening, and remained stable during the night, until early morning. June, August and October were the months of highest density.

A laboratory colony of the sandfly Lutzomyia intermedia was established (at present in its seventh generation) in order to provide specimens for experimental infection and for experimental xenodiagnosis of natural infections due to cutaneous leishmanias in Rio de Janeiro. Date are presented on the methodology and on the yield and duration of each stage of development, during the first four generations.

In a focus of cutaneous leishmaniasis in Jacarepaguá, Rio de Janeiro, one specimen of Lutzomyia intermedia was found naturally infected with Leishmania braziliensis.

To improve our knowledge on the breeding and behaviour of sandflies in captivity, we established closed colonies of Lutzomyia intermedia and Lutzomyia longipalpis. Data are here presented on the feeding preferences of larvae and adults and their influence on the development and survival of each species. Fish food is accepted by the larvae of both species; it is easily available, unexpensive and does not encourage the growth of fungi. The larvae of both species, in all stages accepted food of vegetal and mixed origin, but the 1st and 2nd stage larvae of L. intermedia preferred vegetal food, while the 3rd and 4th stage larvae of L. longipalpis showed a discreet preference for mixed food. Previous feeding on sugar was not necessary to induce a blood meal. Both species can feed on man, dog, hamster and bird, but better development was obtained when the females was fed on hamster. The female of L. longipalpis were more resistant to the absence of blood meal than those of L. intermedia, although 70% of both were able to survive on a sugar meal up to seven days.

Dissection of 765 sandflies captured in Além Paraíba (the type locality of Leishmania braziliensis) resulted in the isolation, from Psychodopygus hirsuta hirsuta, of a parasite of the Le. braziliensis complex.

In Vargem Grande, an endemic area of cutaneous leishmaniasis in State of Rio de Janeiro, where Lutzomyia intermedia had been found infected with Leishmania braziliensis, we performed a series of captures of sandflies to increase the knowledge on their behaviour. The following species were found among 8,671 sandflies collected: L. intermedia, L. migonei, L. longipalpis, L. lanei, L. fischeri, L. firmatoi, L. monticola, L. schreiberi, L. whitmani, L. pelloni, L. barrettoi e L. (Pi) sp. Inside houses and outdoors close to the houses, on human bait and in light traps, the predominant species was L. intermedia, followed by L. migonei, both being more numerous between 9 and 11 p.m. than from 6 to 8 p.m. In a plantation, L. migonei was predominant. In simultaneous captures on man and dog as baits, L. intermedia was more frequent on the former, L. migonei on the latter.

As a preparatory stage for a study aiming at identifying the species and subspecies of local Leishmania in naturally infected sandflies through immunoradiometric assay with monoclonal antibodies, we tried to obtain experimental infections of phlebotomines with well characterized stocks of parasites, in order to test the effectiveness of the method.