Brain cell karyotype of the phlebotomine sand fly Lutzomyia shannoni (Dyar) (Diptera: Psychodidae)

The brain cell karyotype of New World sand fly Lutzomyia shannoni was described. This species has four pairs of chromosomes, 2N=8, with one pair of heteromorphic chromosomes.     

Seasonality of Lutzomyia fairtigi (Diptera: Psychodidae: Phlebotominae), a species endemic to Eastern Colombia

The bionomics of sand flies (Diptera: Phlebotominae) was studied monthly for two consecutive years in alluvial gallery forests in the department of Casanare, Northeastern Colombia. A total of 2,365 specimens and 10 species were captured using CDC light traps and Shannon traps, and from diurnal resting places, and human landing collections. Lutzomyia fairtigi Martins (55%), Lutzomyia micropyga (Mangabeira) (20.9%), and Lutzomyia antunesi (Coutinho) (13.5%) were the predominant species in the region. Lutzomyia flaviscutellata and Lutzomyia panamensis, potential vectors of Leishmania in Colombia and neighboring countries, were also collected, but in low numbers. Lu. fairtigi is an endemic species to Colombia, and minimal data are available on its biology and distribution. The present study provides additional information about Lu. fairtigi, such as the diurnal activity displayed by females on cloudy days, the greater density during the rainy season (April to October), marked anthropophilia, and the presence of flagellates in the midgut of one female.     

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.     

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.     

A first record of Phlebotominae from Argentinean Patagonia (Diptera: Psychodidae: Phlebotominae)

A species of the Lutzomyia oswaldoi group is recorded from the Valcheta stream basin, Somuncura plateau, Patagonia. It represents the World southermost record of a species of Phlebotominae, apparently supporting the hypothesis about the relictual character of several components of the Somuncura plateau biota, particularly for those species belonging to the Paranaense lineages.     

Ultrastructural cytochemistry of the sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae: Phlebotominae)

The sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae) were analyzed by cytochemical techniques. In adult males, the epithelium at the fourth abdominal tergite is modified into a glandular epithelium, with large columnar gland cells located side by side. The gland cell cytoplasm contains a large number of mitochondria and peroxisomes, the latter with positive (electron-dense) reaction for catalase, a typical peroxisomal enzyme marker. The gland cell cytoplasm also contains a central vacuolated area, with a large number of electron-lucent vacuoles, not limited by a unit membrane. In well-preserved preparations such vacuoles present a homogenous and slightly electron-dense content, typical of lipid droplets. Indeed, incubation of the tergites with imidazole-buffered osmium tetroxide (to detect lipids) resulted in positive reaction in these vacuoles, as well as in between the microvilli of the gland cells. Use of the osmium–potassium iodide (Os–KI) technique allowed to demonstrate the presence of several endoplasmic reticulum (ER) profiles, as expected in secretory cells. Our data suggest that ER, lipid droplets and peroxisomes are involved in the sand fly pheromone biosynthesis.     

Phlebotominae (Diptera: Psychodidae) of human leishmaniosis sites in Tunisia

In order to identify the phlebotomine sandfly populations in Tunisian leishmaniosis foci, an entomological survey was carried out through three entomological seasons (2002-2003-2004) in 19 visceral and cutaneous leishmaniosis areas, located in six bioclimatic zones. Sandfly collections were based on light and sticky traps placed around human leishmaniosis cases. 8,722 phlebotomine sandflies belonging to 12 species were collected. The dominance of subgenus Larroussius species in northern foci, Phlebotomus papatasi in south-western foci and their co-dominance in the centre of the country is in accordance with the distribution of Leishmania infantum and L. major in Tunisia. The low density found in the historical zoonotic cutaneous leishmaniosis focus of Metlaoui in the south-west may indicate the high competence of the local populations. Studied phlebotomine settlements have showed a low specific diversity in most of the studied sites. In L. infantum areas, the dominant species were respectively: P. perfiliewi in the cutaneous leishmaniosis site of the humid bioclimatic stage, P. perniciosus in the cutaneous and visceral leishmaniosis foci of semi-arid and arid bioclimatic stages and P. longicuspis in the visceral leishmaniosis focus of saharan bioclimate. In the zoonotic cutaneous leishmaniosis foci, P. papatasi was a dominant species. In the well-known south-eastern foci of cutaneous leishmaniosis due to L. killicki, P. sergenti was a dominant species with P. perniciosus. In the central emerging foci of L. killicki, P. perniciosus was a dominant species in some sites whereas it was very rare in others. In these sites, the subgenus Paraphlebotomus was always present with a higher abundance of P. alexandri than P. sergenti.     

Experimental comparison of anthropophily between geographically dispersed populations of Lutzomyia whitmani (Diptera: Psychodidae)

Lutzomyia whitmani, a major vector of cutaneous leishmaniasis in Brazil, occupies diverse habitats from the Amazon forest canopy to suburban animal pens. Three mitochondrial lineages of Lu. whitmani ('Amazonian', 'North-South' and 'North-east') have parapatric distributions coinciding with different ecological zones. We assessed the host preferences of populations representing the three lineages in standardized field experiments, and found that Lu. whitmani in all sites were significantly more attracted to humans than to dogs or chickens. Females from a southerly population of the North-South lineage showed the greatest degree of anthropophily. Lu. whitmani from Amazonia were also strongly attracted to human baits, contradicting previously published accounts. Intraspecific comparisons in non-Amazonian sites suggest that Lu. whitmani is less anthropophilic than Lu. intermedia but more so than Lu. longipalpis. No significant difference was detected in anthropophily between Lu. whitmani in the Amazon and either Lu. dendrophyla or Lu. gomezi. Anthropophilic behaviour was demonstrated in the same site for Lu. complexa, Lu. flaviscutellata and Lu. brachyphalla, but not for Lu. infraspinosa.     

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.     

Moonlight and blood-feeding behaviour of Lutzomyia intermedia and Lutzomyia whitmani (Diptera:Psychodidae:Phlebotominae), vectors of American cutaneous leishmaniasis in Brazil

Lutzomyia intermedia (Lutz & Neiva, 1912) and L. whitmani (Antunes & Coutinho, 1939) (Diptera: Psychodidae: Phlebotominae), two important vectors of American cutaneous leishmaniasis in Brazil, occur in sympatry in the locality of Posse county, Petropolis municipality, state of Rio de Janeiro, Brazil. We investigated the influence of the lunar cycle on the frequency of specimens of the two species caught while attempting to bite the collectors and in CDC light traps. Analysis of the numbers of sand flies captured in different lunar phases for two consecutive years in the peridomestic site and forest shows that there is a significant positive correlation between moonlight intensity and the numbers of L. intermedia and L. whitmani females collected while blood-feeding, whereas the opposite was observed for the CDC traps.     

Sugars in the alimentary canal of Lutzomyia youngi (Diptera: Phlebotominae)

Abstract. Gas chromatography (GC) analysis of 159 specimens (144 females and 15 males) of Lutzomyia youngi collected in Shannon traps in a coffee plantation in the Andean region of western Venezuela, where leishmaniasis is endemic, revealed the presence of fructose, sucrose, glucose and maltose in the gut and crop of the wild sandflies. The identification of the sugars was confirmed by comparing retention times with those observed for standard sugars and those obtained from sandflies experimentally fed on known sugar solutions. Although the sandflies in nature may ingest each of the four sugars, the results suggest that it is more probable there is an invertase enzyme (glycosidase?) in the gut or crop of the sandfly which hydrolyses ingested disaccharides (e.g. sucrose) to the constituent monosaccharides (i.e. fructose and glucose). Ecological and altitudinal distributions of sandfly species may be related to the availability of specific sugar sources, with epidemiological implications. Identification of the preferred sugar could make breeding easier and would facilitate further research on Leishmania–wector relationships.     

DNA Barcoding for the Identification of Sand Fly Species (Diptera,Psychodidae, Phlebotominae) in Colombia

Sand flies include a group of insects that are of medical importance and that vary in geographic distribution, ecology, and pathogen transmission. Approximately 163 species of sand flies have been reported in Colombia. Surveillance of the presence of sand fly species and the actualization of species distribution are important for predicting risks for and monitoring the expansion of diseases which sand flies can transmit. Currently, the identification of phlebotomine sand flies is based on morphological characters. However, morphological identification requires considerable skills and taxonomic expertise. In addition, significant morphological similarity between some species, especially among females, may cause difficulties during the identification process. DNA-based approaches have become increasingly useful and promising tools for estimating sand fly diversity and for ensuring the rapid and accurate identification of species. A partial sequence of the mitochondrial cytochrome oxidase gene subunit I (COI) is currently being used to differentiate species in different animal taxa, including insects, and it is referred as a barcoding sequence. The present study explored the utility of the DNA barcode approach for the identification of phlebotomine sand flies in Colombia. We sequenced 700 bp of the COI gene from 36 species collected from different geographic localities. The COI barcode sequence divergence within a single species was <2% in most cases, whereas this divergence ranged from 9% to 26.6% among different species. These results indicated that the barcoding gene correctly discriminated among the previously morphologically identified species with an efficacy of nearly 100%. Analyses of the generated sequences indicated that the observed species groupings were consistent with the morphological identifications. In conclusion, the barcoding gene was useful for species discrimination in sand flies from Colombia.     

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.     

Carbohydrate digestion in Lutzomyia longipalpis' larvae (Diptera - Psychodidae)

Lutzomyia longipalpis is the principal species of phlebotomine incriminated as vector of Leishmania infantum, the etiological agent of visceral leishmaniasis in the Americas. Despite its importance as vector, almost nothing related to the larval biology, especially about its digestive system has been published. The objective of the present study was to obtain an overview of carbohydrate digestion by the larvae. Taking in account that phlebotomine larvae live in the soil rich in decaying materials and microorganisms we searched principally for enzymes capable to hydrolyze carbohydrates present in this kind of substrate. The principal carbohydrases encountered in the midgut were partially characterized. One of them is a α-amylase present in the anterior midgut. It is probably involved with the digestion of glycogen, the reserve carbohydrate of fungi. Two other especially active enzymes were present in the posterior midgut, a membrane bound α-glucosidase and a membrane bound trehalase. The first, complete the digestion of glycogen and the other probably acts in the digestion of trehalose, a carbohydrate usually encountered in microorganisms undergoing hydric stress. In a screening done with the use of p-nitrophenyl-derived substrates other less active enzymes were also observed in the midgut. A general view of carbohydrate digestion in L. longipalpis was presented. Our results indicate that soil microorganisms appear to be the main source of nutrients for the larvae.     

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.