Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis

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.

Genetic variability among populations of Lutzomyia (Psathyromyia) shannoni (Dyar 1929) (Diptera: Psychodidae: Phlebotominae) in Colombia

Polyacrylamide gel electrophoresis was used to elucidate genetic variation at 13 isozyme loci among forest populations of Lutzomyia shannoni from three widely separated locations in Colombia: Palambí (Nariño Department), Cimitarra (Santander Department) and Chinácota (Norte de Santander Department). These samples were compared with a laboratory colony originating from the Magdalena Valley in Central Colombia. The mean heterozygosity ranged from 16 to 22%, with 2.1 to 2.6 alleles detected per locus. Nei's genetic distances among populations were low, ranging from 0.011 to 0.049. The estimated number of migrants (Nm=3.8) based on Wright's F-Statistic, F ST, indicated low levels of gene flow among Lu. shannoni forest populations. This low level of migration indicates that the spread of stomatitis virus occurs via infected host, not by infected insect. In the colony sample of 79 individuals, the Gpi locus was homozygotic (0.62/0.62) in all females and heterozygotic (0.62/0.72) in all males. Although this phenomenon is probably a consequence of colonization, it indicates that Gpi is linked to a sex determining locus.

Ootaxonomic investigation of five Lutzomyia species (Diptera, Psychodidae) from Venezuela

The eggshell fine structure of five sand fly species from Venezuela belonging to the genus Lutzomyia (L. migonei, L. ovallesi, L. absonodonta, L. gomezi and L. panamensis) was examined by scanning electron microscopy. The chorionic sculpturing of L. migonei, L. ovallesi, L. absonodonta and L. gomezi was characterized by series of columns arranged in palisade to form sinuous ridges. In inter-ridge areas, the basal layer was covered with fibrous material. The outer chorion of L. panamensis had a pattern known as "mountain- or volcano-like". The morphology of the posterior pole and aeropyle had a common structure in the five species, with some species-specific characters. The eggshell features of the five species are compared with those of other phlebotomine sand flies.

Effect of a second bloodmeal on the oesophagus colonization by Leishmania mexicana complex in Lutzomyia evansi (Diptera: Psychodidae)

Migration and colonization of the oesophagus by Leishmania mexicana parasites were enhanced after digestion of a second bloodmeal intake in Lutzomyia evansi. This event has epidemiological significance since it affects the infection susceptibility of this sand fly species, which is a proven vector of L. chagasi in Colombian and Venezuelan visceral leishmaniasis foci. Also, it may explain the host seeking behaviour displayed by some partially bloodfed flies found inside houses.

First report of Lutzomyia (Nyssomyia) umbratilis Ward & Frahia, 1977 outside of Amazonian Region, in Recife, State of Pernambuco, Brazil (Diptera: Psychodidae: Phlebotominae)

Lutzomyia umbratilis, a known vector of Leishmania guyanensis in the north of Amazon basin, has been exclusively found in the Amazonian region. Here we report for the first time the occurrence of this species in northeastern Brazil. The epidemiological importance of the occurrence of this species in the Atlantic Forest is commented.

A new sand fly, Lutzomyia campograndensis sp. n. (Diptera: Psychodidae: Phlebotominae) from the State of Mato Grosso do Sul, Brazil

During studies of the phlebotomine sand fly fauna of Campo Grande, State of Mato Grosso do Sul, Brazil, a new species was captured with CDC light trap and is described here. The new species resembles Lutzomyia lutziana (Costa Lima) and is named Lutzomyia campograndensis sp. n. after the type locality.

Effect of Lutzomyia whitmani (Diptera: Psychodidae) salivary gland lysates on Leishmania (Viannia) braziliensis infection in BALB/c mice

Previous reports showed that Lutzomyia longipalpis saliva exacerbate Leishmania braziliensis infection in mice. The sand fly Lu. whitmani is one of the vectors of L. (Viannia) braziliensis (LVb), a causative agent of cutaneous leishmaniasis in the State of Ceará, Brazil. To determine whether saliva of Lu. whitmani could increase the infectivity of LVb in mice, we inoculated groups of BALB/c mice with LVb promastigotes in the presence or absence of the salivary glands lysate from Lu. whitmani. We found that coinjection with Lu. whitmani saliva increased size but not longevity of cutaneous LVb lesions in BALB/c mice, since the formed lesions gradually resolved. The mechanism(s) by which Lu. whitmani saliva might exacerbate LVb infection in BALB/c mice is speculated.

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.

Lutzomyia aldafalcaoae sp. n. a new species of Phlebotominae (Diptera: Psychodidae) from Mato Grosso do Sul, Brazil

A new sand fly species, Lutzomia aldafalcaoae is described from males collected in Vila Trindade, an urban district in Aquidauana county, State of Mato Grosso do Sul, Brazil, in October 1996. Taxonomic remarks and a description of the new species is presented.

Deltamethrin-impregnated dog collars have a potent anti-feeding and insecticidal effect on Lutzomyia longipalpis and Lutzomyia migonei

Deltamethrin-impregnated PVC dog collars were tested to assess if they were effective in protecting dogs from sand fly bites of Lutzomyia longipalpis and Lu. migonei. A protective effect against Old World species Phlebotomus perniciosus was demonstrated before. Four dogs wearing deltamethrin collars and three dogs wearing untreated collars (not impregnated with deltamethrin) were kept in separate kennels for over eight months in a village on the outskirts of Fortaleza in Ceará, Brazil. Periodically, a dog from each group was sedated, placed in a net cage for 2 h in which 150 female sand flies had been released 10-15 min before. Lu. longipalpis were used 4, 8, 12, 16, 22, 27, and 35 weeks after the attachment of the collars. Lu. migonei were used 3, 7, 11, 15, 22, 26, and 36 weeks after attachment. During 35 weeks, only 4.1% (81 of 2,022) Lu. longipalpis recovered from the nets with the deltamethrin collared dogs were engorged, an anti-feeding effect of 96%. Mortality initially was over 90% and at 35 weeks was 35% with half of the sand flies dying in the first 2 h. In contrast, 83% of the 2,094 Lu. longipalpis recovered from the nets containing the untreated collared dogs were engorged and the mortality ranged from zero to 18.8% on one occasion with 1.1% dying in the first 2 h. Similar findings were found with Lu. migonei: of 2,034 sand flies recovered over this period, only 70 were engorged, an anti-feeding effect of 96.5%, and mortality ranged from 91% initially to 46% at 36 weeks. In contrast, engorgement of controls ranged from 91 to71% and a mortality ranged from 3.5 to 29.8%. These studies show that deltamethrin impregnated collars can protect dogs against Brazilian sand flies for up to eight months. Thus, they should be useful in a program to control human and canine visceral leishmaniasis.

Characteristics of the bological cycle of Lutzomyia evandroi Costa Lima & Antunes, 1936 (Diptera: Psychodidae) under experimental conditions

Lutzomyia evandroi Costa Lima and Antunes, 1936 is found in Rio Grande do Norte, northeastern Brazil, in areas of visceral and mucocutaneous leishmaniasis and follows the same geographic distribution of L. longipalpis. The biological cycle, oviposition, morphological and behavioral characteristics of the species were studied under experimental conditions. The average number of eggs per wild caught female varied from 21 to 50 eggs along the year, with a peak occurring between January and March and another in August, with oviposition lasting for 4 to 12 days. The mean larval phase was 24 days. Ovipositing rates were influenced by rainfall and temperature indexes, with an increase of eggs per oviposition at the beginning and at the end of the rainy season, and a decrease at the peak of the rainy season.

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.

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.

Frequency of Infection of Lutzomyia Phlebotomines with Leishmania braziliensis in a Brazilian Endemic Area as Assessed by Pinpoint Capture and Polymerase Chain Reaction

Leishmania infected of Lutzomyia spp. are rare in endemic areas. We tested the hypothesis that there is clustering of infected vectors by combining pinpoint capture with sensitive L. braziliensis kDNA minicircle specific PCR/dot blot in an endemic area in the State of Bahia. Thirty out of 335 samples (10 to 20 sand flies/sample; total of 4,027 female sand flies) were positive by PCR analysis and dot blot leading to a underestimated overall rate of 0.4% positive phlebotomines. However, 83.3% of the positive samples were contributed by a single sector out of four sectors of the whole studied area. This resulted in a rate of 1.5% Leishmania positive phlebotomines for this sector, far above rates of other sectors. Incidence of American cutaneous leishmaniasis cases for this sector was about twice that for other sectors. Our results show that there is a non-homogeneous distribution of Leishmania-infected vectors. Such a clustering may have implications in control strategies against leishmaniasis, and reinforces the necessity of understanding the ecological and geographical factors involved in leishmanial transmission.

Redescription of the Male of Lutzomyia lutziana (Costa Lima) and Description of the Female (Diptera: Psychodidae)

During the course of taxonomic studies of New World phlebotomine sand flies, it was discovered that the female specimen considered to be the female of Lutzomyia lutziana does not belong to this species. A new description of the female of L. lutziana is presented, based on specimens captured in Lassance, State of Minas Gerais, the type locality of the species. The male of L. lutziana is also redescribed, based on an exemplar caught in Curvelo, State of Minas Gerais.