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The Cecropia genus is widely distributed in Latin America including at least 60 species, and some of them are commonly used in traditional medicine for the treatment of several diseases. We used Cecropia pachystachya Trécul to search for quorum sensing (QS) inhibitors compounds and found that the aqueous extract of C. pachystachya leaves is a promising source of substances with this activity. Using as biosensor Chromobacterium violaceum ATCC 31532 and Escherichia coli pSB403, the compounds chlorogenic acid (2), isoorientin (3), orientin (4), isovitexin (6), vitexin (7), and rutin (9) were identified as QS inhibitors. None of these compounds inhibited the growth of neither the used biosensors nor the microorganisms Staphylococcus aureus ATCC 23591, Escherichia coli ATCC 25922 and Saccharomyces cerevisiae, used here as growth inhibition controls. Along with the rutin, here we presented for the first time the QS-inhibition potential of the C-glycosyl flavonoids. The prospective of this evidence lead to the use of these compounds as antipathogenic drugs or antifoulants.

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Canine visceral leishmaniasis (VL), caused by Leishmania infantum (Leishmania chagasi in the New World), is a zoonotic, endemic disease in Western Europe and Latin America. The potential spreading to new regions was suggested by the appearance of canine VL among foxhounds in the US. Although the sand fly vectors in the major foci of transmission have been described, no information exists on other sand flies that could propagate the infection outside endemic areas. We evaluated the capacity of Lutzomyia shannoni (Dyar) and Lutomyia youngi (Feliciangeli & Murillo), which are widely distributed in the New World, to acquire L chagasi (Cunha and Chagas) infections. A high proportion of L youngi were infected after feeding on an oligosymptomatic dog (51 per cent) or a polysymptomatic individual (95 per cent), but the intensity of infection was low (< 200 promastigotes/fly). L shannoni became infected only by feeding on the polysymptomatic dog, and the infection rate was lower (9 per cent) than in Lutzomyia longipalpis (36 per cent), and Lutzomyia evansi (Nunez-Tovar) (Lutz and Neiva) (38 per cent), but the intensity of infection (200 to > 500 promastigotes/fly) was comparable (L longipalpis) or higher (L evansi) than in the New World vectors. It is hypothesised that the presence of infected dogs in areas where L shannoni or L youngi occur could initiate new endemic cycles of VL in both South and North America.

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We studied the reservoir competency of canines with distinct clinical presentations of Leishmania chagasi infection. The parasitologic status of asymptomatic and symptomatic dogs was determined by standard culture methods Infectivity was assessed by multiple xenodiagnoses with Lutzomyia longipalpis, over a period of 2-11 months. Asymptomatic dogs were non-infective (0 of 5) while 2 of 7 oligosymptomatic dogs infected L longipalpis, transmitting the parasites at low rates (range 0.9-5.2% of engorged flies). Polysymptomatic dogs transmitted L. chagasi more frequently (4 of 8 dogs) and reached higher infection rates (range 5.0-22.5% of engorged flies). The skin of the ear tended to be more infective to sand flies than that of the abdomen. Polymerase chain reaction hybridization (PCR-H) was a sensitive method for detection of L. chagasi, yielding the highest positive rate in serum (16 of 17 dogs) with no distinction between clinical groups. No association between skin positivity by PCR-H and infectivity to sand flies was found. The infectivity of dogs from clinically comparable groups from Colombian and Mediterranean foci differed. This may be a reflection of varied nutritional conditions or vector competency of distinct sand fly species.

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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.

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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.

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During field studies of enzootic Venezuelan equine encephalitis (VEE) viruses associated with epizootic emergence, a large number of virus isolates were made in sylvatic foci of Venezuela and Colombia. To rapidly characterize these isolates, antigenic subtypes were determined by means of immunofluorescence and by single-strand conformational polymorphism (SSCP) analysis by use of an 856-bp fragment from the P62 gene, which we used to distinguish genetic variants. Representative isolates were sequenced to assess the sensitivity of SSCP to detect genetic differences. The SSCP analysis distinguished isolates differing by as little as 1 nucleotide; overall, differences of > or = 1 nucleotide were recognized 89% of the time, and the sensitivity to distinguish strains that differed by only 1 or 4 nucleotides was 17 and 57%, respectively. Phylogenetic analyses of representative sequences showed that all recent isolates from the Catatumbo region of western Venezuela and the middle Magdalena Valley of Colombia were closely related to epizootic subtype IAB and IC strains; strains from Yaracuy and Miranda States were more distantly related. Cocirculation of the same virus genotype in both Colombian and Venezuelan foci indicated that these viruses are readily transported between enzootic regions separated by > 300 km. The SSCP analysis appears to be a simple, fast, and relatively efficient method of screening VEE virus isolates to identify meaningful genetic variants.

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Hexamerins are proteins found in high abundance in the haemolymph of larval and adult insects. The expression patterns of the genes encoding the house fly, Musca domestica, hexamerins were determined by Northern analyses using cDNAs as probes. A cDNA, A1, hybridized to a fat body-specific messenger RNA (mRNA) which is detectable in larvae until pupation. Antibodies raised to the larval-specific hexamerin, Hex-L, bind recombinant protein encoded by a 5' rapid amplification of cDNA ends (RACE) product of A1, A2, indicating that the A cDNAs likely represent the genes encoding Hex-L. The F1, F2 and F3 cDNAs, corresponding to genes encoding an adult, female-enriched hexamerin, Hex-F, hybridized with an mRNA isolated from protein-fed females which has a temporal expression profile similar to that observed for the accumulation of Hex-F. Furthermore, expression of the mRNAs hybridizing to the F cDNAs is correlated with the abundance of Hex-F protein during the gonotrophic cycles. The mRNA transcription profiles indicate that the Hex-L and Hex-F genes are regulated in a sex-, tissue- and developmental phase-dependent manner. This stage-specific expression of hexamerins contrasts with the expression patterns of hexamerins seen in other insects. The conceptual translation products of larval hexamerin cDNAs showed identity with larval serum protein 1 (LSP1)-type hexamerins while the deduced products of the female hexamerin cDNAs showed the highest identity with LSP2-type hexamerins. Genomic analyses showed that the larval hexamerin and female hexamerin genes from M. domestica belong to two distinct multigenic families.

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Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28 masculineC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.

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Adult Anopheles darlingi salivary glands are paired organs located on either side of the esophagus. The male glands consist of a single small lobe. The female gland is composed of two lateral lobes, with distinct proximal and distal portions, and a medial lobe. The lobes are acinar structures, organized as a unicellular epithelium that surrounds a salivary canal. The general cellular architecture is similar among the lobes, with secretory material appearing as large masses that push the cellular structures to the periphery of the organ. Cells of the proximal-lateral lobes show asynchronous cycles of secretory activity and contain secretory masses with finely filamentous aspect. In the distal-lateral lobes, cells display synchronous cycles of activity, and have a dense secretory product with mottled pattern. Cells of the medial lobe have secretory masses uniformly stained and highly electrondense. Biochemical analysis of the adult female salivary glands revealed apyrase, alpha-glucosidase and lysozyme activities. Alpha-glucosidase and lysozyme activities are detected mostly in the proximal lobes while apyrase is mainly accumulated in the distal lobes. This differential distribution of the analyzed enzymes reflects a specialization of different regions for sugar and blood feeding. Thus, the morphological differences observed in the lobes correlate with functional ones.

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Recent studies have indicated that epizootic Venezuelan equine encephalitis (VEE) viruses can evolve from enzootic, subtype ID strains that circulate continuously in lowland tropical forests (A. M. Powers, M. S. Oberste, A. C. Brault, R. Rico-Hesse, S. M. Schmura, J. F. Smith, W. Kang, W. P. Sweeney, and S. C. Weaver, J. Virol. 71:6697-6705, 1997). To identify mutations associated with the phenotypic changes leading to epizootics, we sequenced the entire genomes of two subtype IC epizootic VEE virus strains isolated during a 1992-1993 Venezuelan outbreak and four sympatric, subtype ID enzootic strains closely related to the predicted epizootic progenitor. Analysis by maximum-parsimony phylogenetic methods revealed 25 nucleotide differences which were predicted to have accompanied the 1992 epizootic emergence; 7 of these encoded amino acid changes in the nsP1, nsP3, capsid, and E2 envelope glycoprotein, and 2 were mutations in the 3' untranslated genome region. Comparisons with the genomic sequences of IAB and other IC epizootic VEE virus strains revealed that only one of the seven amino acid changes associated with the 1992 emergence, a threonine-to-methionine change at position 360 of the nsP3 protein, accompanied another VEE virus emergence event. Two changes in the E2 envelope glycoprotein region believed to include the major antigenic determinants, both involving replacement of uncharged residues with arginine, are also candidates for epizootic determinants.

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Baseline biological growth data of Lutzomyia shannoni (Dyar) were compared under two experimental conditions within insulated styrofoam chests and in standard laboratory incubators. The developmental time from egg to adult was 67 and 52 days, respectively. Based on cohorts of 100 females in each experiment, horizontal life tables were constructed. The following predictive parameters were obtained under each of the two conditions: net rate of reproduction (23.5 and 18.0 females per cohort female), generation time (11.4 and 9.4 weeks), intrinsic rate of population increase (0.27 and 0.30), and finite rate of population increment (1.31 and 1.36). The reproductive value for each class age of the cohort females was calculated. The observed parameters were obtained under each experimental condition: net rate of reproduction (1.9 and 2.5 females per cohort female), generation time (11.7 and 9.6 weeks), intrinsic rate of population increase (0.05 and 0.09), and finite rate of population increment (1.06 and 1.10). Vertical life tables were elaborated and mortality was described for every generation in each cohort. In addition, for two successive generations, additive variance and heritability for fecundity were estimated.

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The life cycle of Lutzomyia shannoni (Dyar), was described for laboratory conditions with maximum daily temperature of 27-30 degree C, minimum daily temperatures of 22-27 degree C and relative humidity between 87-99%. Life cycle in each stage was as follows: egg 6-12 days (ave, 8.5 days); first stage larva 5-13 days (ave. 9.6 days); second stage larva 4-13 days (ave. 9.2 days); third stage larva 5-19 days (ave. 11.8 days); fourth stage larva 7-37 days (ave. 19.9 days); pupa 7-32 days (ave. 15.2 days). The life expectancy of adults ranged from 4 to 15 days (ave. 8.6 days). The entire egg to adult period ranged from 36 to 74 days (ave. 54.6 days). On average, each female oviposited 22.7 eggs; the average egg retention per female was 24.3 eggs.

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A cDNA encoding a lysozyme expressed specifically in the salivary glands of the malaria vector mosquito, Anopheles darlingi, was isolated by differential screening an adult female salivary gland library with abdomen and salivary gland cDNAs. The primary nucleic acid sequence of the cDNA contains a deduced coding region of 429 nucleotides and 5'- and 3'-end non-transcribed regions. A signal peptide of twenty-three amino acids and a mature protein of 120 amino acids are evident in the conceptual translation product. The results of RT-PCR experiments indicated that in adult mosquitoes this gene is expressed specifically in the salivary glands. Lysozyme enzymatic activity was detected in the salivary glands and abdomens of adult mosquitoes, but the pH optimum differed for each tissue and this was interpreted to indicate the presence of more than one enzyme, each being expressed in a different tissue. The salivary gland lysozyme may be involved in protection against bacterial infection in the anterior portion of the mosquito digestive tract.

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Lutzomyia longipalpis (Lutz & Neiva), the sand fly vector of American visceral leishmaniasis in the New World tropics, has a broad but discontinuous geographical distribution from southern Mexico to Argentina. A baseline for population genetic structure and genetic variability for this species was obtained by analyzing 5 local, peridomestic populations at the approximate center of its distribution, the Magdalena River Valley of central Colombia. Three populations of L. longipalpis from El Callejón, a small rural community, were compared with 2 populations from neighboring areas 12 and 25 km distant for genetic variation at 15 isoenzyme loci. The mean heterozygosity ranged from 11 to 16%, with 1.2 to 2.3 alleles detected per locus. Nei's genetic distances among the populations were very low, ranging from 0.001 to 0.007. Gene flow estimates based on FST indicated high levels of gene flow among local L. longipalpis populations, with minimal population substructuring.

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An intensive search for the larval habitats of Lutzomyia longipalpis (Lutz & Neiva) was conducted from November 1992 to October 1993 at a small rural community in Colombia where American visceral leishmaniasis is endemic. Emergence traps constructed from polyvinyl chloride pipes were used to sample a variety of soil microhabitats that included edge areas of covered pigpens, cattle corrals, the base of trees, and leaf litter at sites within 40 m of a house, rocks in fields located between 50 and 500 m from houses, and sites within a patch of secondary forest (rocks, base of palm trees, and leaf litter). The teneral status of the sand flies captured in the emergence traps was confirmed by laboratory studies that determined the rate of terminalia rotation in male L. longipalpis and the rate of cuticular growth layer formation of the thoracic phragma in both sexes of this species. A total of 58 teneral sand flies was captured during the study period (49 wk). Fifteen specimens were L. longipalpis; of these 11 (5 sand flies per square meter) were captured near pigpens, 3 (1.4 sand flies per square meter) were captured near rock resting sites, and 1 (1.6 sand flies per square meter) was collected at the base of a tree. The remainder of the sand flies were either L. trinidadensis (Newstead) or L. cayennensis (Flock & Abonnenc). Our results indicate that L. longipalpis larvae were dispersed widely in sites near houses, rather than concentrated in a few optimal microhabitats.

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Lutzomyia longipalpis, 15 other species of the genus Lutzomyia, and one species of Brumptomyia were collected in an endemic focus of cutaneous leishmaniasis in a river canyon 450 m above sea-level, in Rio Claro, Antioquia, Colombia. The presence of Lu. longipalpis is associated with the destruction of the primary forest and the development of new farmland and rural settlement in this region. The composition of species identified a different habitat for Lu. longipalpis in Colombia. Lu. yuilli and Lu. longipalpis were predominant (68.26%) followed by Lu. trapidoi, Lu. hartmani, Lu. triramula, Lu. panamensis, Lu. gomezi.

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A simple device for marking phlebotomine sand flies with fluorescent powders is described and tested; the design of the new device is characterized by separate compartments for sand flies and powder. The effect of fluorescent powder on survival and mobility of the sand fly Lutzomyia longipalpis was compared using our device (method A) and a single-container method (method B). Mortality within 1 h of powder application was negligible for method A (0.9%), but was 19.8% for method B; in addition, method B also reduced sand fly mobility. Adherence of excess powder to the sand flies observed with method B was responsible for the negative effects observed during the marking process. In field releases, however, recapture rates were the same for each method. Neither sand fly mobility or survival were adversely affected if appropriate quantities (method A) of fluorescent powder were applied to the exoskeleton of these insects.

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Ecological studies on the sand fly Lutzomyia longipalpis (Lutz & Neiva) were conducted during 1990-1992 in a small rural community in Colombia where American visceral leishmaniasis (AVL) is endemic. Subsamples of sand flies collected weekly from pigpens, the interior of houses, and natural outdoor resting sites were dissected to determine physiological age and Leishmania chagasi Cunha & Chagas infection rates. Eleven female L. longipalpis had flagellates in their gut, 2 of which were successfully cultured and identified as Leishmania chagasi. The reproductive status, stage of ovarian development, and trophic history of female sand flies varied among sites, habitats, and time of collection. The percentage of parous females ranged from about one-third to two-thirds overall and varied seasonally. Of most relevance to AVL transmission was the finding that 8% of L. longipalpis females were multiparous. In addition, our data suggest that L. longipalpis rest inside houses after blood-feeding outdoors, and that this species can blood-feed more than once during a single gonotrophic cycle.

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Nocturnal activity of the sand fly Lutzomyia longipalpis (Lutz & Neiva) was studied from August 1991 to July 1992 in a small rural community in Colombia where American visceral leishmaniasis is endemic. During 2 or 3 nights each month, sand flies were collected with hand-held aspirators each hour between 1730 and 0630 hours, from a pigpen and a cattle corral located 30 m apart. Host-seeking activity of L. longipalpis adults was characterized by 2 general patterns: (1) adult sand fly activity increased shortly after sunset and continued until just after sunrise, and (2) peak sand fly activity was greatest early in the evening (1830-2330 hours) and then declined steadily toward morning. Female L. longipalpis activity generally increased after 2030 hours, whereas that of males remained constant or declined as the evening progressed. There were seasonal differences in sand fly abundance between the 2 sites: peak abundance in the cattle corral occurred during hot, dry periods, whereas maximum abundance in the pigpen occurred when relative humidity was higher. Influence of relative humidity on activity varied with season. Sand fly activity tended to decrease at temperatures below 24 degrees C and increase in the presence of moonlight.

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