Studies on populations of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in Brazil

Studies were performed on five Brazilian populations of Lutzomyia longipalpis: Salvaterra (PA), S?o 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.     

Report of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in a cutaneous-leishmaniasis-endemic area of Panama

Lutzomyia longipalpis is the primary vector of the parasite responsible for visceral leishmaniasis in the Americas. In the present study, Lu. longipalpis was found in a domiciliary area in Limón, a district in Capira, a region in which cutaneous leishmaniasis is endemic in Panama. Previously, this species has been found in a humid forest in this same region. Finding Lu. longipalpis in domiciliary areas indicates that this species may be adapting to new habitats and that it may play a role in the transmission of leishmaniasis in Panama.     

Biology of Lutzomyia intermedia Lutz & Neiva, 1912 and Lutzomyia longipalpis Lutz & Neiva, 1912 (Diptera, Psychodidae), under experimental conditions. I. Feeding aspects of larvae and adults

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.     

Variations in the gonostyle of Nyssomyia intermedia (Lutz & Neiva) (Diptera: Psychodidae)

This paper describes sand flies similar to Nyssomyia intermedia (Lutz & Neiva) with variations in the number of spines at the gonostyle and tests the hypothesis whether these specimens belong or not to N. intermedia species. Using Principal Component Analysis and Neighbour Joining, the measurements of 15 structures of the phlebotomine with variations in number of spines were compared with measurements of 30 sand flies of N. intermedia species. Both analyses didn't cluster the specimens with variation in spines number in one group and provided evidence to the hypothesis that those sand flies belong to N. intermedia.     

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 Maranh?o 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 Maranh?o, 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.     

Establishment of a colony, in laboratory, of Lutzomyia intermedia Lutz & Neiva, 1912 (Diptera, Psychodidae, Phlebotominae)

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.     

Ultrastructural features of the midgut epithelium of females Lutzomyia intermedia (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae).

A morphological study of the midgut of Lutzomyia intermedia, the primary vector of cutaneous leishmaniasis, in southeast Brazil, was conducted by light, scanning and transmission electron microscopy. The midgut is formed by a layer of epithelium of columnar cells on a non-cellular basal lamina, under which there is a musculature, which consists of circular and longitudinal muscular fibers. A tracheolar network is observed surrounding and penetrating in the musculature. Females were examined 12, 24, 48, 72 h and 5 days following a blood meal and were analyzed comparatively by transmission electron microscopy with starved females. In starved females, the epithelium of both the anterior and posterior sections of the midgut present whorl shaped rough endoplasmic reticulum. The posterior section does not present well-developed cellular structures such as mitochondria. Observations performed at 12, 24, 48 and 72 h after the blood meal showed morphological changes in the cellular structures in this section, and the presence of the peritrophic matrix up to 48 h after the blood meal. Digestion is almost complete and a few residues are detected in the lumen 72 h after blood feeding. Finally, on the 5th day after the blood meal all cellular structures present the original feature resembling that seen in starved sand flies. Morphometric data confirmed the morphological observations. Mitochondria, nuclei and microvilli of midgut epithelial cells are different in starved and blood fed females. The mitochondria present a similar profile in the epithelium of both the anterior and posterior section of the midgut, with higher dimension in starved females. The cell microvilli in the posterior section of the midgut of starved females are twice the size of those that had taken a blood meal. We concluded that there are changes in the midgut cellular structures of L. intermedia during the digestion of blood, which are in agreement with those described for other hematophagous diptera.     

Stomodaeal valve ultrastructure in the sandfly Lutzomyia longipalpis (Diptera: Psychodidae)

The stomodaeal valve and adjacent cardia region of the alimentary canal of the phlebotomine sandfly Lutzomyia longipalpis have been studied using electron microscopy. With the scanning electron microscope (SEM), numerous pit-like structures of different sizes were found in the surface of the valve of male and female flies. Under the transmission electron microscope (TEM), the cells adjacent to the pores were shown to have numerous mitochondria and to lack neural innervation. It is suggested that the pit-like structures are probably secretory. A typical basiconic sensilla was found on the inner side of the oesophagus at the junction with the stomodaeal valve, with at least one dendrite which extended to the tip, indicating a chemosensory role. It is likely that this type of sensilla may control the movement of the stomodaeal valve after contact with fluids entering the mid-gut and may be responsible for diverting the sugar meal to the crop by closure of the valve.     

Ingestion of saliva during carbohydrate feeding by Lutzomyia longipalpis (Diptera; Psychodidae)

The aim of this study was to obtain experimental evidence that phlebotomine saliva is actually ingested during the carbohydrate ingestion phase (before and after blood digestion). The ingestion of carbohydrate was simulated as it occurs in the field by offering the insects balls of cotton soaked in sucrose, sucrose crystals or orange juice cells. The results obtained here showed that ingestion occurred under each condition investigated, as indicated by the presence of apyrase, an enzyme used as a marker to detect saliva in the insect gut and/or carbohydrate sources. Saliva ingestion by phlebotomine during the carbohydrate ingestion phase is important to explain how it could promote starch digestion and to trigger Leishmania promastigotes to follow a differentiation pathway as proposed previously by some authors.     

The effect of male sandfly pheromone and host factors as attractants for female Lutzomyia longipalpis (Diptera: Psychodidae)

Abstract Male Lutzomyia longipalpis (Lutz & Neiva) pheromone extracted from tergal glands combined with heat, carbon dioxide gas and hamster urine was found to be attractive to virgin female sandflies. The host factors if offered in the absence of pheromone or a heat source were, however, unattractive to female flies. Pheromone stored for 6 days and then combined with the same host components remained attractive to female flies but storage failed to enhance its attractiveness. The effects of carbon dioxide gas, hamster urine, chicken uropygial gland extract and octenol plus acetone on the responses of females were inconsistent. A ceramic battery powered heat source was tested with pheromone and host attractants and showed potential for field application in developing a pheromone trap.     

Speciation and population structure in the morphospecies Lutzomyia longipalpis (Lutz & Neiva) as derived from the mitochondrial ND4 gene

Recent studies have suggested that the phlebotomine sand fly Lutzomyia longipalpis (Diptera: Psychodidae), the principal vector of visceral leishmaniasis in the Neotropics, may consist of several allopatric sibling species. Phylogenetic and population genetic analyses of nucleotide variation in a 618-bp fragment of the mitochondrial ND4 gene were carried out on specimens of Lu. longipalpis from several locations in Central and South America. The analyses were concordant with previous findings, indicating that certain allopatric populations of Lu. longipalpis have become sufficiently differentiated as to represent sibling species. Phylogenetic analyses revealed deep genetic divisions between four clades represented by specimens from northern South America, Brazil, Central America, and an isolated Colombian population. Strong differentiation also was observed between certain populations within the first two clades. Partitioning of genetic diversity within and between Central American populations did not show the presence of more than one species in the region. However, distance, even within the 70-km range of the Honduran collection sites, was found to have a remarkably strong effect on gene flow. The highly subdivided population structure may be due to the patchiness of their distribution. F(ST) values comparing a Guatemalan population with several Honduran populations revealed a level of differentiation associated with a negligible rate of gene flow.     

Navigation of Lutzomyia longipalpis (Diptera: Psychodidae) under dusk or starlight conditions

The responses of male and female Lutzomyia longipalpis (Lutz & Neiva) to different wavelengths of light was tested by presenting the sandflies with two light sources simultaneously, a series of test wavelengths between 350-670 nm and a 400 nm control. To test whether L. longipalpis could discriminate between the test and control, three sets of experiments were carried out in which the test wavelengths were presented at higher, equivalent or lower intensity than the control. In all three experiments, ultra-violet (350 nm) and blue-green-yellow (490-546 nm) light was more attractive to L. longipalpis than the control wavelength. However, at low intensity, UV was less attractive, than equivalent or higher intensity UV light. At intensities equivalent to or higher than the control wavelength, ultra-violet light was more attractive than blue-green. Furthermore, at low intensity, green-yellow (546 nm) light was more attractive to males whereas blue-green (490 nm) was more attractive to females. Blue-violet (400 nm) and orange-red (600-670 nm) light were least attractive in all three sets of experiments. Response function experiments indicated that the responses were dependent on both intensity and wavelength and that therefore more than one photoreceptor must be involved in the response. The results indicated that L. longipalpis can discriminate between different wavelengths at different intensities and thus have true colour vision. It also suggests that L. longipalpis may be able to navigate at dusk or under moonlight or starlight conditions using light in the blue-green-yellow part of the spectrum. The difference in response of males and females to light in this region is interesting and may indicate the different ecology of the sexes at night. Overall, these results may have important implications for sandfly trap design.     

Development of Leishmania (Viannia) braziliensis vianna, 1911 in Lutzomyia intermedia (Lutz & Neiva, 1912) (Diptera:Psychodidae:Phlebotominae) under experimental conditions.

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.