World Sand Fly Research Articles

Genetic diversity of ribosomal RNA internal transcribed spacer sequences in Lutzomyia species from areas endemic for New World cutaneous leishmaniasis

In this study, each of 60 rRNA internal transcribed spacer (ITS) 1 and ITS2 sequences was determined from 44 individuals of 14 morphologically identified New World sand fly Lutzomyia species in Ecuador, and their interspecies and intraspecies genetic diversity was compared. Distinguishing between related species based on the ITS1 sequence was difficult because of variability, while the genetic diversity of ITS2 was distinct even among closely related species. Further, an assessment of intraspecies ITS sequence diversity in the subgenus Helcocyrtomyia revealed no correlation between sequence variation and geographic distribution. The results strongly suggested ITS2 to be a more suitable marker than ITS1 for the taxonomic analysis of Lutzomyia species including closely related species. Moreover, neither ITS sequence may be useful for the analysis of population structures in Lutzomyia species.

EST sequencing of blood-fed and Leishmania-infected midgut of Lutzomyia longipalpis, the principal visceral leishmaniasis vector in the Americas

Leishmaniasis is an important worldwide public health problem. Visceral leishmaniasis caused by Leishmania infantum chagasi is mainly transmitted by Lutzomyia longipalpis in the Americas. Leishmania development within the sand fly vector is mostly restricted to the midgut. Thus, a comparative analysis of blood-fed versus infected midguts may provide an invaluable insight into various aspects of sand fly immunity, physiology of blood digestion, and, more importantly, of Leishmania development. To that end, we have engaged in a study to identify expressed sequenced tags (ESTs) from L. longipalpis cDNA libraries produced from midguts dissected at different times post blood meal and also after artificial infection with L. i. chagasi. A total of 2,520 ESTs were obtained and, according to the quality of the sequencing data obtained, assembled into 378 clusters and 1,526 individual sequences or singletons totalizing 1,904 sequences. Several sequences associated with defense, apoptosis, RNAi, and digestion processes were annotated. The data presented here increases current knowledge on the New World sand fly transcriptome, contributing to the understanding of various aspects of the molecular physiology of L. longipalpis, and mechanisms underlying the relationship of this sand fly species with L. i. chagasi.

Constitutive and blood meal‐induced trypsin genes in Lutzomyia longipalpis

Trypsins constitute some of the most abundant midgut digestive proteases expressed by hematophagous insects upon blood feeding. In addition to their role in the digestion of the blood meal, these proteases also have been implicated in the ability of certain pathogens to infect their natural vector. In sand flies, digestive proteases including trypsins were associated with early killing of Leishmania and are believed to play a role in the species-specificity dictating sand fly vectorial capacity. Our group is involved in studies of midgut digestive proteases in the sand fly Lutzomyia longipalpis, the principal vector of visceral leishmaniasis in Brazil. Here we report on the identification of two cDNAs, Lltryp1 and Lltryp2, which code for putative midgut trypsins in L. longipalpis. Analyses of RNA abundance using semi-quantitative RT-PCR show a different pattern of expression between the two genes. Lltryp1 expression remains undetected until blood feeding and reaches a peak at 12 h post-blood meal (PBM), returning to pre-blood meal levels at 72 h PBM. Additionally, Lltryp1 expression is undetected during larval development. Lltryp2, on the other hand, is constitutively expressed as high levels in the non-blood fed female, but is reduced upon blood feeding. At the end of the digestive cycle, Lltryp2 regains its pre-blood meal levels. This cDNA also is present in all developmental stages and in adult males. This pattern of expression is reminiscent of what is seen in mosquitoes and Old World sand flies, but has characteristics that are unique to L. longipalpis.

Detection of species-specific antibody response of humans and mice bitten by sand flies

Sand fly saliva plays an important role in Leishmania transmission. We characterized the host antibody response to saliva from 3 sand fly species. Specific IgG was observed in sera from experimentally bitten mice as well as in sera from individuals living in the endemic area of Leishmania tropica in Sanliurfa, Turkey. Sera of Sanliurfa inhabitants showed high IgG levels against saliva of Phlebotomus sergenti and P. papatasi, the 2 most abundant sand fly species in this area, but did not react with saliva of the New World sand fly, Lutzomyia longipalpis. Patients with active Le. tropica lesions possessed significantly higher anti-P. sergenti IgG levels than the healthy individuals from the same place while anti-P. papatasi IgG levels were equal in both groups. Major protein bands in P. papatasi and P. sergenti saliva reacted with both, human and mice sera; in P. papatasi, however, mouse IgG recognized preferentially the 42 kDa protein band while the human IgG reacted strongly with the 30 kDa band. Our data suggest that the antibody response to sand fly saliva could be used for monitoring the exposure of humans and other hosts to sand flies and might be used as a marker of risks for Leishmania transmission in endemic areas.

INFLAMMATORY CELL INFILTRATION AND HIGH ANTIBODY PRODUCTION IN BALB/c MICE CAUSED BY NATURAL EXPOSURE TO LUTZOMYIA LONGIPALPIS BITES

Sand flies inject saliva into the mammalian host when probing for a blood meal. Understanding the initial vertebrate reactions against sand fly saliva is important for possible interventions because these insects transmit diseases to humans and other animals. Little is known of these reactions to New World sand flies. Repeated exposure of BALB/c mice to Lutzomyia longipalpis bites leads to local inflammatory cell infiltration comprised of neutrophils, macrophages, and eosinophils. Total IgG and IgG1 antibodies react predominantly with three major protein bands (45, 44, and 16 kD) of the insect saliva by Western blot. The injection of immune serum previously incubated with salivary gland homogenate induced an early infiltration with neutrophils and macrophages, suggesting the participation of immune complexes in triggering inflammation.

Correlation of male genital filaments and female spermathecal ducts in New World sand flies of the Lutzomyia intermedia species complex (Diptera: Psychodidae, Phlebotominae).

The lengths of the male genital filaments and female spermathecal ducts were measured in phlebotomine sand flies of the Lutzomyia intermedia species complex and the ratios between these characters calculated. Ratios for L. intermedia s. s. from Northeast vs Southeast Brazil (Espírito Santo and Minas Gerais), Espírito Santo/Minas Gerais vs Rio de Janeiro/São Paulo and L. intermedia vs L. neivai were significantly different at P < 0.1, 0.05 and 0.01 respectively when compared using ANOVA. The spermathecal ducts and genital filaments of L. intermedia were significantly longer than those of L. neivai (P < 0.01) and could be used to differentiate these species. The taxonomic and biological significance of these differences is discussed.

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.

Expression of Heterologous Promoters in &lt;I&gt;Lutzomyia longipalpis&lt;/I&gt; and &lt;I&gt;Phlebotomus papatasi&lt;/I&gt; (Diptera: Psychodidae) Cell Lines

To establish a transient expression system for genes introduced into sand fly cell lines, we tested the expression of the luciferase reporter gene under control of different promoters. Towards this end, we lipofected cell lines obtained from New and Old World sand flies, LL-5 from Lutzomyia longipalpis Lutz & Neiva and PP-9 from Phlebotomus papatasi Scopoli, respectively. The relative levels of luciferase expression were studied under control of Drosophila melanogaster Meigen heat shock protein 70 (hsp70), human cytomegalovirus, simian virus 40 or Junonia coenia (Hübner) densovirus (P9) promoters. The Drosophila heat shock protein 70 promoter, originating from insect genes, functioned as a strong promoter in both cell lines. Promoters from the different virus genes also were capable of driving transgene expression in both cell lines.

Species Composition of the Phlebotomine Sandfly Fauna (Diptera: Phlebotominae) in Mokolo Region, Northern Cameroon

Two studies were undertaken to determine the species composition of the phlebotomine sandfly fauna in the Mokolo region, an endemic focus of cutaneous leishmaniasis in northern Cameroon. The preliminary survey conducted in July 1994 using sticky paper traps recorded 843 sandfly specimens, all belonging to the two genera of the Old World sandflies, Sergentomyia and Phlebotomits. The species included S. (Sergentomyia) schzvetzi, S. (Sergentomyia) antennata, S. (Parrotomyia) africana and P. (Phlebotomus) duboscqi: From January 1996 to December 1996, a total of 3241 sandflies were collected using sticky paper traps. In this sampling, six out of the total of eight species recorded were of the genus Sergentomyia. In addition to the species recorded in the preliminary study, S. (S.) bedfordi, S. (Grassomyia) squamipleuris, S. (Sintonius), clydei and P. (Anaphlebotomus) rodhaini were sampled in 1996. This is the first report of S. squamipleuris ir Cameroon. The implications of this study to the transmission of cutaneous leishmaniasis are discussed with particular reference to P. duboscqi, a known vector of the disease in the region.

Quantitative morphological evidence for incipient species within Lutzomyia quinquefer (Diptera: Psychodidae).

Morphological variation among geographic populations of the New World sand fly Lutzomyia quinquefer (Diptera, Phlebotominae) was analyzed and patterns detected that are probably associated with species emergence. This was achieved by examining the relationships of size and shape components of morphological attributes, and their correlation with geographic parameters. Quantitative and qualitative morphological characters are described, showing in both sexes differences among local populations from four Departments of Bolivia. Four arguments are then developed to reject the hypothesis of environment as the unique source of morphological variation: (1) the persistence of differences after removing the allometric consequences of size variation, (2) the association of local metric properties with meristic and qualitative attributes, rather than with altitude, (3) the positive and significant correlation between metric and geographic distances, and (4) the absence of a significant correlation between altitude and general-size of the insects.

Interspecific hybridization and genetic variability of Phlebotomus sandflies.

The first successful hybridization is reported between Phlebotomus papatasi and P. duboscqi, two important Old World sandfly vectors of leishmaniasis and other diseases. Laboratory strains of P. papatasi and P. duboscqi were separable by six diagnostic enzyme loci: Est-3, Idh-1, Mdh-2, Mpi, Tre-1 and Tre-3. Hybrids between the two species were verified by the recovery of heterozygous isozyme patterns for the diagnostic loci. No F2 or backcross progeny were obtained. P. papatasi was separated from P. bergeroti by three diagnostic enzyme loci: Est-3, Mpi and Pgd. The isozyme patterns of P. bergeroti contain elements of both P. duboscqi and P. papatasi, although seven diagnostic loci (Est-3, Idh-1, Me, Mpi, Pgd, Tre-1 and Tre-3) separated P. bergeroti from P. duboscqi. Genetic variability profiles of the three species were established for 20 enzyme loci. Three geographically distant strains of P. papatasi from Calcutta, Maharashtra and Israel had isozyme genetic distances of < 0.05. The recently established Calcutta strain showed an unexpectedly low genetic variability with only one (Idh-2) of 20 loci being polymorphic (average heterozygosity of 1.9%) in contrast to 5-8 polymorphic loci (10-12% heterozygosity) in the Maharashtra and Israel strains. Mass and single pair crosses between the three P. papatasi strains were fertile with normal progeny numbers. Thus we found no signs of speciation in P. papatasi.

Phlebotomus papatasiSand Fly Salivary Gland Lysate Down-Regulates a Th1, but Up-Regulates a Th2, Response in Mice Infected withLeishmania major

AbstractA vertebrate host becomes infected with Leishmania major when the sand fly vector injects parasites into skin along with saliva. Previous studies showed that salivary gland lysate of the New World sand fly Lutzomyia longipalpis markedly enhanced L. major infection in CBA mice. However, L. major is an Old World parasite transmitted in nature by the Old World sand fly Phlebotomus papatasi. Here we examine the ability of P. papatasi salivary gland lysate to enhance infection (lesion size and parasite burden) by L. major. In addition, we examine the effects of salivary gland lysate on the immune response to L. major by monitoring the levels of cytokine mRNA from the lymph nodes draining cutaneous lesions. We found that P. papatasi salivary gland lysate dramatically exacerbated lesion development in disease-resistant CBA mice. This exacerbation of disease correlated with inhibition of the production of Th1 cytokines and associated factors (IFN-γ, IL-12, and inducible nitric oxide synthase), but with enhancement of the Th2 cytokine IL-4, whereas no changes in the levels of IL-10 and TGF-β were noted. Importantly, salivary gland lysate directly up-regulated expression of IL-4 mRNA in mice in the absence of infection with L. major.

Peritrophic envelopes of Lutzomyia spinicrassa (Diptera: Psychodidae).

The secretion, morphology, and chemical composition of the peritrophic envelope were studied in the phlebotomine sand fly, Lutzomyia spinicrassa Morales, OsornoMesa, Osorno & Hoyos, a suspected vector of Leishmania braziliensis in Colombia and Venezuela. Viewed under light microscopy, the envelope matured rapidly and could be dissected from the blood bolus as early as 12 h and until 36 h after feeding; subsequently it began to degrade. The envelope was initially a closed sac around the blood meal, but opened posteriorly in most flies by 6 h. The posterior opening may facilitate the migration and establishment of Le. braziliensis in the hindgut. Secretion of envelope precursors was from the entire midgut epithelium. Electron microscopy revealed that electron-dense precursor material (possibly chitin) was present, bathing the microvilli during the first 12 h after blood feeding. This secretion appeared to originate from the bases of the microvilli. From 1 to 36 h, an electron-lucid precursor material (possibly protein) was secreted from the entire length of microvilli and from their bases. Both precursors appeared to be formed at the epithelial surface, not associated with secretory vesicles. The envelope developed rapidly from precursor material, and by 6 h a defined electron-lucid structure was present above the microvilli. Most mature envelopes (12-36 h) were 0.5-2.1 microns thick, multilayered, wholly electron-lucid, and composed of microfibrils and granules. Electron-dense components were seen in some envelopes at 24-36 h. An anterior hyaline plug was present from 12 to 36 h. Envelopes were composed of chitin, protein, and glycoprotein, based on chemical and histochemical tests. The likely presence of several amino acids (lysine, aspartic acid, and glutamic acid) that may cross-link chitin and protein was demonstrated by a positive ninhydrin-Schiff test. This study constitutes the first ultrastructural investigation of peritrophic envelope development by a New World sand fly.

Life Cycle of Leishmania major (Kinetoplastida: Trypanosomatidae) in the Neotropical Sand Fly Lutzomyia longipalpis (Diptera: Psychodidae)

The development of Leishmania major Yakimoff & Schokhor in the New World sand fly Lutzomyia longipalpis (Lutz & Neiva) was examined by light and electron microscopy. In this unnatural host, parasites differentiated into 10 typical morphological forms, multiplied at three sites, migrated anteriorly and established in the foregut, and attached to gut surfaces. In the blood meal, amastigotes divided and transformed into two successive dividing, stumpy promastigote stages. Elongate nectomonad promastigotes developed from stumpy forms and subsequently rounded up in some flies into paramastigotes and opisthomastigotes. Differentiation into round opisthomastigotes and the apparent fusion of paramastigotes in the blood meal were novel observations in this study. Three nectomonad promastigotes--elongate, short, and metacyclic--were free-swimming in the midgut lumen. Elongate nectomonad promastigotes were highly oriented in the midgut, with their flagella embedded between the epithelial microvilli. Short haptomonad promastigotes were the predominant form attached to the intima of the stomodeal valve, whereas pear-shaped haptomonad promastigotes and paramastigotes colonized surfaces of the esophagus and pharynx. Peripylarian attachment of promastigotes and paramastigotes in the pylorus, ileum, and colon was noted in 21% of flies, suggesting that suprapylarian leishmanias have not lost the ability to colonize the hindgut. L. longipalpis was a successful biological host for L. major, allowing complete development of the parasite.

Dispersal of the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) at an endemic focus of visceral leishmaniasis in Colombia.

Mark-release-recapture studies were carried out during 1990-1991 in El Callejón, Colombia, an endemic focus of American visceral leishmaniasis, to study the longevity, dispersal, and flight range of the principal vector, Lutzomyia longipalpis Lutz & Neiva. Several groups of wild-caught (n = 1,539) and laboratory-reared F1 (n = 2,208) sand flies were marked with fluorescent dusts and released. Recaptures at daytime resting sites, on animal bait, and in CDC light traps were made for 20 d following each release. From 2 to 9% of L. longipalpis were recaptured, a rate which differed between the sexes (7.7% male, 1.5% female). Overall, 49% of the L. longipalpis were recaptured between 0 and 50 m, 48% within 100 and 300 m, and nearly 3% at > or = 0.5 km from the release site. Sex differences in recapture site, distance flown, and direction were observed. Our results indicate that the dispersal behavior of peridomestic populations of L. longipalpis compares more closely with that of Old World sand fly species from similar habitats than to the sylvan Neotropical phlebotomines.

A new microsporidian parasite, Flabelliforma montana n.g., n.sp., infecting Phlebotomus ariasi (Diptera, Psychodidae) in France

Flabelliforma montana, a new microsporidian genus and species, is described from the sandfly Phlebotomus ariasi. The parasite was found in sandflies collected in the commune of Roquedur, Gard, France, and represents the first microsporidium recorded from Old World sandflies. Meronts lie in direct contact with the host cell cytoplasm and divide by binary fission or plasmotomy. Sporogonial plasmodia divide by multiple fission within a sporophorous vesicle, passing through a lobed fan-like stage before division into uninucleate sporoblasts. All stages have unpaired nuclei. Transmission is direct. Flabelliforma is one of nine genera of microsporidia with multisporous sporogony, all but one of which are currently considered to belong to one family, Pleistophoridae, but in reality are not closely related. The characters which differentiate Flabelliforma from the other multisporous genera are discussed and a key is provided for identification of the genera.

Ultrastructural Comparison of Egg Surface Morphology of Five Lutzomyia Species (Diptera: Psychodidae)

Surface structure of sand fly eggs of five species are illustrated with scanning electron micrographs. Eggs studied were from the following New World sand fly species: Lutzomyia anthophora (Addis), Lutzomyia cruciata (Coquillett), Lutzomyia diabolica (Hall), Lutzomyia shannoni (Dyar), and Lutzomyia vexator (Coquillett). Eggs of these five species and of 36 previously described species were grouped into four morphological categories based on egg surface patterns: polygonal, connected parallel ridges, unconnected parallel ridges, and volcanolike. Several methods used for fixation of sand fly eggs are discussed.

Laboratory testing of repellents against the sand fly Phlebotomus papatasi (Diptera: Psychodidae).

Eight topical repellents and a synthetic pyrethroid were evaluated against the Old World sand fly Phlebotomus papatasi , using a dose-response testing procedure on white rabbits. The decreasing order of repellent effectiveness based on the median effective dosages was: Citronyl; MGK 11; permethrin; Indalone; N,N-diethyl-m-toluamide; MGK 326; 2-ethyl-l,3-hexanediol; dibutyl phthalate; and dimethyl phthalate. The sensitivity of P. papatasi to repellents is similar to that of Lutzomyia longipalpis .

Psychodopygus as a Genus: Reasons for a Flexible Classification of the Phlebotomine Sand Flies (Diptera: Psychodidae)

The ranking of Psychodopygus as a genus rather than a subgenus is substantiated by a comparative analysis of the principal characters distinguishing the world's sand fly genera. Reviews of previous classifications and the characters employed in them show that the wide divergence of opinion among sand fly taxonomists has not been reconciled by a recently proposed “stable” classification that, for New World species, is considered unacceptable for the following reasons. Accepting the use of one “central,” or principal, character for differentiating genera will inhibit the search for critical combinations of taxonomic characters that reflect the evolutionary paths taken by neotropical sand flies; and, the curtailment of systematic studies can only weaken the impetus to compare and relate different sand fly species, thereby hindering the discovery of new information of possible zoological and medical importance.