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.     

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.     

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.     

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.     

Correlates of the peridomestic abundance of Lutzomyia longipalpis (Diptera: Psychodidae) in Amazonian Brazil

Abstract. Abundance of the sandfly Lutzomyia longipalpis , the vector of Leishmania chagasi , was surveyed using CDC light-suction traps in fifteen villages and 180 homesteads on Marajo island, Para State, Brazil. Flies were most abundant in cultivated areas, secondary growth and open woodland, away from savanna grassland and forest. Within homesteads, the abundance of flies in animal sheds was weakly associated with the number of hosts kept therein, and inversely related to the number elsewhere, both human and animal. Houses harboured more flies if dogs were present and if the house had: a roof of thatch rather than of tiles or tin; a thatched roof which had not been treated with insecticide; relatively more holes in walls and roof. The sex ratio of flies was more male-biased at higher densities. We weigh the importance of these variables as risk factors for Le.chagasi infection.     

Wing geometry as a tool for studying the Lutzomyia longipalpis (Diptera: Psychodidae) complex

Toro Toro (T) and Yungas (Y) have been described as genetically well differentiated populations of the Lutzomyia longipalpis (Lutz & Neiva, 1912) complex in Bolivia. Here we use geometric morphometrics to compare samples from these populations and new populations (Bolivia and Nicaragua), representing distant geographical origins, qualitative morphological variation ("one-spot" or "two-spots" phenotypes), ecologically distinct traits (peridomestic and silvatic populations), and possibly different epidemiological roles (transmitting or nor transmitting Leishmania chagasi). The Nicaragua (N) (Somotillo) sample was "one-spot" phenotype and a possible peridomestic vector. The Bolivian sample of the Y was also "one-spot" phenotype and a demonstrated peridomestic vector of visceral leishmaniasis (VL). The three remaining samples were silvatic, "two-spots" phenotypes. Two of them (Uyuni and T) were collected in the highlands of Bolivian where VL never has been reported. The last one (Robore, R) came from the lowlands of Bolivia, where human cases of VL are sporadically reported. The decomposition of metric variation into size and shape by geometric morphometric techniques suggests the existence of two groups (N/Y/R, and U/T). Several arguments indicate that such subdivision of Lu. longipalpis could correspond to different evolutionary units.     

Molecular and Behavioral Differentiation among Brazilian Populations of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae)

Background

Lutzomyia longipalpis is the primary vector of American visceral leishmaniasis. There is strong evidence that L. longipalpis is a species complex, but until recently the existence of sibling species among Brazilian populations was considered a controversial issue. In addition, there is still no consensus regarding the number of species occurring in this complex.

Methodology/Principal Findings

Using period, a gene that controls circadian rhythms and affects interpulse interval periodicity of the male courtship songs in Drosophila melanogaster and close relatives, we analyzed the molecular polymorphism in a number of L. longipalpis samples from different regions in Brazil and compared the results with our previously published data using the same marker. We also studied the male copulation songs and pheromones from some of these populations. The results obtained so far suggest the existence of two main groups of populations in Brazil, one group representing a single species with males producing Burst-type copulation songs and cembrene-1 pheromones; and a second group that is more heterogeneous and probably represents a number of incipient species producing different combinations of Pulse-type songs and pheromones.

Conclusions/Significance

Our results reveal a high level of complexity in the divergence and gene-flow among Brazilian populations of the L. longipalpis species complex. This raises important questions concerning the epidemiological consequences of this incipient speciation process.     

Midgut pH profile and protein digestion in the larvae of Lutzomyia longipalpis (Diptera: Psychodidae)

The sand fly Lutzomyia longipalpis is the vector of Leishmania infantum, the etiological agent of American visceral leishmaniasis. Despite its importance, until now the internal anatomy of the immature forms has never been described and little is known about their digestive processes. In nature, sand fly larvae feed on organic detritus in the soil, constantly ingesting large amounts of material. The objective of this study was to describe the anatomy of the gut and the pH of the gut lumen, as well as to investigate the proteases responsible for protein digestion. The larvae have a short gut with a prominent, well-developed midgut. Ingestion of food containing indicator dyes permitted the gut pH to be measured. A pH gradient was observed, varying from >9 in the anterior midgut to 6.5-7.0, in the posterior midgut. The endoproteolytic enzymes are secreted in the anterior midgut and are able to digest azocasein over a large pH range, specially at pH 11. Studies with various inhibitors indicated that the digestive endoproteases are trypsin- and chymotrypsin-like enzymes. These results were confirmed by using the substrates BApNA and N-CBZ-L-PpNA, specific for trypsin and chymotrypsin, respectively. Aminopeptidases were also investigated with p-nitroaniline-derived substrates. These enzymes are located in the posterior midgut, bound to the membranes and functioning at an optimal pH of 6.5-8.0. The results presented here are consistent with the current proposal that proteins are digested to peptides in the anterior midgut inside the endoperitrophic space and subsequently undergo digestion in the ectoperitrophic space of the posterior midgut.     

Sugar feeding and fluid destination control in the phlebotomine sandfly Lutzomyia longipalpis (Diptera: Psychodidae)

Sandfly feeding behaviour and destination of coloured sugar meals in the gut of Lutzomyia longipalpis were investigated with particular attention to the role of the crop. Sandflies were able to ingest sugar from liquid drops, microcapillaries, a slice of pear and even sugar powder. In most cases the flies adopted a 'sugar feeding mode' with raised palps. As the fruit dried, flies of both sexes fed by piercing the tissue with the proboscis. All sugar-fed flies had a full crop plus a small amount of sugary fluid in the thoracic mid-gut, i.e. past the stomodaeal valve. Dissections of flies interrupted during feeding showed that the very first trace of sugar passed through the stomodaeal valve, but that the rest of the meal was diverted into the crop. This suggests that closure of the stomodaeal valve is initiated only after a small volume of sugar solution has passed through it.     

Host-parasite relationships of Ascogregarina chagasi (Eugregarinorida, Aseptatorina, Lecudinidae) in Lutzomyia longipalpis (Diptera: Psychodidae)

The life cycle of Ascogregarina chagasi in larvae and in adult female Lutzomyia longipalpis was studied by light and electron microscopy. Sporozoites and young gamonts were attached to the epithelial lining of the larval midgut via an osmiophilic contact zone. The mucron of young gamonts was bordered by an invaginated pellicular fold, and an electron-opaque vesicular structure was observed adjacent to it. Sporozoites possessed an apical complex and were bound by a double membrane with underlying subpellicular microtubules. The gamont pellicle was uniformly corrugated and consisted of two cortical membranes and a plasma membrane. Mature gamonts and gametocysts were found in the posterior ectoperitrophic space of third and fourth instar larval midguts and in the haemocoel of adult flies. Gametocysts in adult females adhered to the genital accessory glands, where they were encased in electron-dense capsules secreted by the fly through haemocyte-mediated humoral immune reactions. Oocytes were spindle-shaped and bound by a double-layered wall with a discernible polar plug at each end. Sporulation was endogenous, occurring within gametocysts in the midguts of larvae or the accessory glands of adult females. FITC-phalloidine staining of all stages of A. chagasi except mature gametocysts produced bright fluorescence, indicating the presence of a diffuse, actin-like protein in the cytoplasm.     

Activity of Lutzomyia pseudolongipalpis and L. longipalpis s.l. (Diptera: Psychodidae) in Venezuela

The nocturnal activity of the phlebotomine sandfly Lutzomyia pseudolongipalpis and two populations of L. longipalpis s.l. from different American visceral leishmaniasis foci in Venezuela was studied using collection bottle rotator traps. The activity of L. pseudolongipalpis from Lara State was continuous throughout the night, while that of L. longipalpis s.l. from El Layero, Guárico State and from Santa Ana del Valle, Margarita Island, was greatest before 23:00 h. The activity of sandflies of both populations and sexes steadily decreased thereafter. These different patterns seem to correlate with genetic data that indicate the presence in Venezuela of at least two sibling species in the L. longipalpis complex. The advantages of the bottle rotator trap for this type of study are discussed.     

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.     

Life tables and reproductive parameters of Lutzomyia spinicrassa (Diptera: Psychodidae) under laboratory conditions

Lutzomyia spinicrassa is a vector of Leishmania braziliensis in Colombia. This sand fly has a broad geographical distribution in Colombia and Venezuela and it is found mainly in coffee plantations. Baseline biological growth data of L. spinicrassa were obtained under experimental laboratory conditions. The development time from egg to adult ranged from 59 to 121 days, with 12.74 weeks in average. Based on cohorts of 100 females, horizontal life table was constructed. The following predictive parameters were obtained: net rate of reproduction (8.4 females per cohort female), generation time (12.74 weeks), intrinsic rate of population increase (0.17), and finite rate of population increment (1.18). The reproductive value for each class age of the cohort females was calculated. Vertical life tables were elaborated and mortality was described for the generation obtained of the field cohort. In addition, for two successive generations, additive variance and heritability for fecundity were estimated.     

Larval Breeding Sites of Lutzomyia longipalpis (Diptera: Psychodidae) in Visceral Leishmaniasis Endemic Urban Areas in Southeastern Brazil

Background

The scarcity of information on the immature stages of sand flies and their preferred breeding sites has resulted in the focus of vectorial control on the adult stage using residual insecticide house-spraying. This strategy, along with the treatment of human cases and the euthanasia of infected dogs, has proven inefficient and visceral leishmaniasis continues to expand in Brazil. Identifying the breeding sites of sand flies is essential to the understanding of the vector''s population dynamic and could be used to develop novel control strategies.

Methodology/Principal finding

In the present study, an intensive search for the breeding sites of Lutzomyia longipalpis was conducted in urban and peri-urban areas of two municipalities, Promissão and Dracena, which are endemic for visceral leishmaniasis in São Paulo State, Brazil. During an exploratory period, a total of 962 soil emergence traps were used to investigate possible peridomiciliary breeding site microhabitats such as: leaf litter under tree, chicken sheds, other animal sheds and uncovered debris. A total of 160 sand flies were collected and 148 (92.5%) were L. longipalpis. In Promissão the proportion of chicken sheds positive was significantly higher than in leaf litter under trees. Chicken shed microhabitats presented the highest density of L. longipalpis in both municipalities: 17.29 and 5.71 individuals per square meter sampled in Promissão and Dracena respectively. A contagious spatial distribution pattern of L. longipalpis was identified in the emergence traps located in the chicken sheds.

Conclusion

The results indicate that chicken sheds are the preferential breeding site for L. longipalpis in the present study areas. Thus, control measures targeting the immature stages in chicken sheds could have a great effect on reducing the number of adult flies and consequently the transmission rate of Leishmania (Leishmania) infantum chagasi.     

Identification of oviposition attractants for the sandfly Lutzomyia longipalpis (Diptera: Psychodidae) in volatiles of faeces from vertebrates

Abstract. Extracts of volatiles from rabbit and chicken faeces preferentially attracted gravid sandflies, Lutzomyia longipalpis (Lutz and Neiva), in an oviposition bioassay. In electrophysiology experiments, the same extracts selectively stimulated two olfactory cells while inhibiting another in ascoid sensilla on the antennae of these flies. Analysis of faeces volatiles by gas chromatography linked to ascoid sensillum recording revealed two early eluting electrophysiologically active components of rabbit faeces. These active compounds were identified in both rabbit and chicken faeces volatile extracts by gas chromatography -mass spectrometry as hexanal and 2-methyl-2-butanol. Hexanal stimulated one cell type and inhibited another, whereas 2-methyl-2-butanol stimulated a third cell type. A 1:l mixture of synthetic hexanal and 2-methyl-2-butanol elicited the same targeted oviposition response from gravid females on the treatment septum of the bioassay as did the total volatile extract of rabbit or chicken faeces.
The monoterpenes α(+)-pinene (plus some optical and positional isomers) and a-terpinene activated a separate cell type, whereas benzaldehyde stimulated the same receptor as hexanal, but with a higher threshold. Furthermore, an olfactory cell selectively tuned to the perception of the male sex pheromone of this species was also found in the ascoid sensillum.     

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37 masculineC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.