Experimental infection of Phlebotomus perniciosus by bioluminescent Leishmania infantum using murine model and artificial feeder

Leishmaniasis is a vector-borne disease that is transmitted by sandflies and caused by obligate intracellular protozoa of the genus Leishmania. In the present study, we carried out a screening on the experimental infection of Phlebotomus pernioucus by bioluminescent Leishmania infantum using murine model and artificial feeder. We developed a real-time polymerase chain reaction (RT-PCR)-based method to determine individually the number of Leishmania promastigotes fed by infected flies. Among 1840 new emerged female sand flies, 428 were fed on the infected mice. After their death, they were analysed individually by RT-PCR. Our results demonstrated just a single Leishmania positive female at sixth day post meal. A total of 1070 female sand flies were exposed in contact with artificial feeder containing the human blood with two different quantities of Leishmania parasites: 2.106/mL and 1.107/mL. A blood meal including 1.107/mL LUC-promastigotes was proposed to 270 females and 75 (28%) flies were engorged. Among them, 44 (59%) were positive by RT-PCR analysis, with a relative average of 50551 Leishmania parasites. In case of blood feeding of females with 2.106/mL promastigotes, 57 out of 800 (7%) females succeed to feed from artificial feeder which 22 (39%) were positive with a relative average of 6487 parasites.

Phlebotomine fanna (diptera: psychodidae) in anarea of fishing tourism in centtral-western Brazil / Fauna flebotomínea (Diptera: Psychodidae) em área de turismo pesqueiro no Centro-Oeste do Brasil

The aim of this study was to identify behavioral aspects of the sandfly fauna of a fishing tourism area in the municipality of Bonito (MS). Monthly captures were undertaken from December 2009 to November 2010, using automatic CDC type light traps, from 18h00 to 06h00, in a forested area, a savannah area, peridomiciles and animal shelters near peridomiciliary areas. Nyssomyia whitmani was the most frequent out of a total of 6,699 specimens collected, belonging to 16 species, followed by Psathyromyia bigeniculata and Lutzomyia longipalpis, found in all the environments investigated, though in their greatest numbers in the animal shelters. Ny. whitmani exhibited its highest frequencies during the dry months, coincident with the fishing season, when the risk of transmission of cutaneous leishmaniasis for tourists and inhabitants increases. Noteworthy was the finding of two species naturally infected by flagellates: Ny. whitmani and Pa. bigeniculata. The local population and visiting tourists should be warned of the threat posed by leishmaniasis and the health authorities alerted to the need for adopting environmental sanitary measures, especially regarding such animal shelters as they seem to provide favorable conditions to the proliferation, maintenance and breeding opportunities of phlebotomines.

O objetivo deste estudo foi identificar aspectos do comportamento da fauna flebotomínea de área de turismo pesqueiro localizada no município de Bonito (MS). Foram realizadas capturas mensais no período de dezembro de 2009 a novembro de 2010, utilizando armadilhas luminosas automáticas do tipo CDC das 18:00h às 6:00h, em matas, área de cerrado, peridomicílios e abrigos de animais próximos a áreas peridomiciliares. De um total de 6.699 espécimes coletados, pertencentes a 16 espécies, Nyssomyia whitmani foi a mais frequente, seguida de Psathyromyia bigeniculata e Lutzomyia longipalpis, encontradas em todos os tipos de ambientes, porém com maior expressão em abrigos de animais. Ny. whitmani apresentou frequências mais elevadas nos meses mais secos, coincidentes com a estação da pesca, o que eleva o risco de transmissão da leishmaniose tegumentar a turistas e moradores da área. Importante ressaltar o encontro de duas espécies naturalmente infectadas por flagelados: Ny. whitmani e Pa. bigeniculata. A população local e turistas devem ser advertidos da ameaça que representam as leishmanioses e as autoridades de saúde alertadas para adoção de medidas de saneamento ambiental, principalmente com relação aos abrigos de animais, que parecem fornecer condições favoráveis para a proliferação, manutenção e reprodução de flebotomíneos.

Geographic distribution of phlebotomine sandfly species (Diptera: Psychodidae) in Central-West Brazil

This study updates the geographic distributions of phlebotomine species in Central-West Brazil and analyses the climatic factors associated with their occurrence. The data were obtained from the entomology services of the state departments of health in Central-West Brazil, scientific collections and a literature review of articles from 1962-2014. Ecological niche models were produced for sandfly species with more than 20 occurrences using the Maxent algorithm and eight climate variables. In all, 2,803 phlebotomine records for 127 species were analysed. Nyssomyia whitmani, Evandromyia lenti and Lutzomyia longipalpis were the species with the greatest number of records and were present in all the biomes in Central-West Brazil. The models, which were produced for 34 species, indicated that the Cerrado areas in the central and western regions of Central-West Brazil were climatically more suitable to sandflies. The variables with the greatest influence on the models were the temperature in the coldest months and the temperature seasonality. The results show that phlebotomine species in Central-West Brazil have different geographical distribution patterns and that climate conditions in essentially the entire region favour the occurrence of at least one Leishmania vector species, highlighting the need to maintain or intensify vector control and surveillance strategies.

Leishmaniasis transmission: distribution and coarse-resolution ecology of two vectors and two parasites in Egypt

Introduction: In past decades, leishmaniasis burden has been low across Egypt; however, changing environment and land use has placed several parts of the country at risk. As a consequence, leishmaniasis has become a particularly difficult health problem, both for local inhabitants and for multinational military personnel. Methods: To evaluate coarse-resolution aspects of the ecology of leishmaniasis transmission, collection records for sandflies and Leishmania species were obtained from diverse sources. To characterize environmental variation across the country, we used multitemporal Land Surface Temperature (LST) and Normalized Difference Vegetation Index (NDVI) data from the Moderate Resolution Imaging Spectroradiometer (MODIS) for 2005-2011. Ecological niche models were generated using MaxEnt, and results were analyzed using background similarity tests to assess whether associations among vectors and parasites (i.e., niche similarity) can be detected across broad geographic regions. Results: We found niche similarity only between one vector species and its corresponding parasite species (i.e., Phlebotomus papatasi with Leishmania major), suggesting that geographic ranges of zoonotic cutaneous leishmaniasis and its potential vector may overlap, but under distinct environmental associations. Other associations (e.g., P. sergenti with L. major) were not supported. Mapping suitable areas for each species suggested that northeastern Egypt is particularly at risk because both parasites have potential to circulate. Conclusions: Ecological niche modeling approaches can be used as a first-pass assessment of vector-parasite interactions, offering useful insights into constraints on the geography of transmission patterns of leishmaniasis. .

Phlebotomus (Legeromyia) multihamatus subg. nov., sp. nov. from Gabon (Diptera: Psychodidae)

During a research project aimed at the study of the Culicinae fauna of Gabon and carried out in the National Park of La Lopé, we captured an unknown sandfly male specimen (genus Phlebotomus) by CDC miniature light trap belonging to a new species for Science. Furthermore, the originality of his genitalia does not allow us to include this species in one of the existing subgenus, thus in this paper we propose the creation of a new subgenus, as Phlebotomus (Legeromyia) multihamatus sp. nov., subg. nov. described from the National Park of La Lopé, through one male captured with CDC miniature light trap. A new species and a new subgenus of sandfly is characterised by a short style with three spines, a paramere wearing a basal hook as well as a basal pouch and the absence of basal lobe on the coxite. The originality of the genitalia of the male gives way to discussion about potential primary homologies between P. multihamatus sp. nov. and Phlebotomus (Abonnencius) fortunatarum, Phlebotomus (Anaphlebotomus) stantoni and Phlebotomus (Euphlebotomus) argentipes, which should be verified for future studies. The discovery of this new species in Gabon must encourage the study of sandflies in this country.

Appraisal of Phlebotomus argentipes habitat suitability using a remotely sensed index in the kala-azar endemic focus of Bihar, India

Visceral leishmaniasis, or kala-azar, is recognised as a serious emerging public health problem in India. In this study, environmental parameters, such as land surface temperature (LST) and renormalised difference vegetation indices (RDVI), were used to delineate the association between environmental variables and Phlebotomus argentipes abundance in a representative endemic region of Bihar, India. The adult P. argentipes were collected between September 2009-February 2010 using the hand-held aspirator technique. The distribution of P. argentipes was analysed with the LST and RDVI of the peak and lean seasons. The association between environmental covariates and P. argentipes density was analysed a multivariate linear regression model. The sandfly density at its maximum in September, whereas the minimum density was recorded in January. The regression model indicated that the season, minimum LST, mean LST and mean RDVI were the best environmental covariates for the P. argentipes distribution. The final model indicated that nearly 74% of the variance of sandfly density could be explained by these environmental covariates. This approach might be useful for mapping and predicting the distribution of P. argentipes, which may help the health agencies that are involved in the kala-azar control programme focus on high-risk areas.

Flebotomíneos de localidades rurais no noroeste do Estado do Paraná, Brasil / Sandflies in rural localities in northwest Paraná State, Brazil

Relatam-se os resultados de coletas de flebotomíneos em seis lotes na zona rural no Município de Japurá, Estado do Paraná, Brasil, mostrando a presença de flebotomíneos no domicílio, peridomicílio e em matas residuais. As coletas de flebotomíneos foram realizadas com armadilhas de Falcão, das 20:00h às 03:00h. Nos lotes 195, 223, 236 e 527 foram feitas três coletas em diferentes períodos, de dezembro de 2007 a janeiro de 2009. Nos lotes 175 e 218, as coletas foram feitas de janeiro a dezembro de 2008. Coletaram-se 8.453 flebotomíneos, com uma média horária (MH) de 155,40 exemplares. Nyssomyia neivai foi a espécie dominante. A maioria dos flebotomíneos foi coletada em abrigos de animais domésticos (MH = 75,91) e nas matas ciliares (MH = 38,45), nos meses mais quentes e chuvosos, num ambiente peridomiciliar com acúmulo de matéria orgânicas e descarte inadequado de águas de uso doméstico, o que mostra a necessidade da limpeza regular do peridomicílio e do afastamento dos abrigos de animais domésticos das residências.

This study reports the results from sandfly captures at six sites in a rural area of Japurá, Paraná State, Brazil, showing the vector's presence indoors, outdoors, and in residual forest. Sandfly captures were performed with Falcão traps from 8:00 AM to 3:00 PM. At sites 195, 223, 236, and 527, three captures were performed at different times from December 2007 to January 2009. At sites 175 and 218, captures were performed from January to December 2008. 8,453 sandflies were captured, with an average of 155.40 specimens per hour. Nyssomyia neivai was the predominant species. Most sandflies were captured in domestic animal shelters (75.91 per hour) and in ciliary forest (38.45 per hour) during the hottest and rainiest months, in the outdoor environment with accumulation of organic matter and inadequate disposal of household wastewater. The study emphasizes the need for regular outdoor cleaning around households and building domestic animal shelters far from residences.

Proven and putative vectors of American cutaneous leishmaniasis in Brazil: aspects of their biology and vectorial competence

The aim of the present review is to give relevant information on aspects of the biology and ecology, including the vectorial competence of Lutzomyia sand fly species suggested as vectors of American cutaneous leishmaniasis in Brazil. The disease, due to Leishmania (Viannia) braziliensis, has been registered in most municipalities in all the Brazilian states and its transmission is associated with more than one sand fly species in each geographical region. A variety of Leishmania species can be found in the Amazon basin, where different epidemiological chains have been detected with the participation of different phlebotomine vectors. Finally, a discussion is presented on some sand fly species found naturally infected by Leishmania, but for which there is as yet no evidence regarding their epidemiological importance.

Functional characterization of a salivary apyrase from the sand fly, Phlebotomus duboscqi, a vector of Leishmania major.

Two transcripts coding for proteins homologous to apyrases were identified by massive sequencing of a Phlebotomus (P.) duboscqi salivary gland cDNA library. The sequence analysis revealed that the amino acids important for enzymatic activity including nucleotidase activity and the binding of calcium and nucleotides were well conserved in these molecules. A recombinant P. duboscqi salivary apyrase was expressed in Escherichia coli and purified. The resulting protein efficiently hydrolyzed ADP and ATP, but not AMP, GDP, CDP or UDP, in a calcium-dependent manner. Further, the recombinant protein inhibited ADP- and collagen-induced platelet aggregation. The results indicated that this salivary protein plays an important role in the blood-feeding process in P. duboscqi. Its unique enzymatic activity makes the salivary apyrase an attractive candidate as a therapeutic agent for the treatment of thrombotic pathologies as well as a reagent for a wide variety of research purposes.

The subgenus Migonemyia Galati 1995 (Diptera, Psychodidae, Phlebotominae), with description of a new species Migonemyia vaniae: a review

The capture of a new species of the subgenus Migonemyia Galati, 1995 (Diptera, Psychodidae, Phlebotominae), Migonemyia vaniae sp. nov. in the Ribeira Valley, state of São Paulo, Brazil, together with the other two species: Mg. migonei (França, 1920) and Mg. rabelloi (Galati & Gomes, 1992) lead us to review this subgenus. The new species was described and illustrated. The genitalia of the two other species were also illustrated and some genital characteristics (number of setae on the gonocoxite tuft, ejaculatory ducts and pump and ducts/pump ratio; and number of setae on the tergite VIII of the females) considered important to differentiate the three species, including five populations of Mg. migonei (from Northeastern, Southeastern, and Southern Brazilian regions and of Peru) were submitted to variance analyses. The Mg. migonei population of Northeastern Brazilian region showed distinct smaller values (P < 0.05) than the other Brazilian populations studied as regarding these characteristics. The capture of both sexes of these three species in sympatry confirms the association between the sexes of Mg. rabelloi, recognised as doubtful when this species was originally described. Identification keys for male and female of the three species are presented.

Cloning and characterization of trypsin- and chymotrypsin-like proteases from the midgut of the sand fly vector Phlebotomus papatasi.

Trypsin and chymotrypsin serine proteases are the main digestive proteases in Diptera midguts and are also involved in many aspects of the vector-parasite relationship. In sand flies, these proteases have been shown to be a potential barrier to Leishmania growth and development within the midgut. Here we describe the sequence and partial characterization of six Phlebotomus papatasi midgut serine proteases: two chymotrypsin-like (Ppchym1 and Ppchym2) and four trypsin-like (Pptryp1-Pptryp4). All six enzymes show structural features typical to each type, including the histidine, aspartic acid, and serine (H/D/S) catalytic triad, six conserved cysteine residues, and other amino acid residues involved in substrate specificity. They also show a high degree of homology (40-60% identical residues) with their counterparts from other insect vectors, such as Anopheles gambiae and Aedes aegypti. The mRNA expression profiles of these six proteases vary considerably: two trypsin-like proteases (Pptryp1 and Pptryp2) are downregulated and one (Pptryp4) upregulated upon blood feeding. The two chymotrypsin-like enzymes display expression behavior similar to that of the early and late trypsins from Ae. aegypti.

Numerical taxonomy of Old World Phlebotominae (Diptera: Psychodidae). 1 - Considerations of morphological characters in the genus Phlebotomus Rondani & Berté 1840

Numerical analyses (correspondence analyses, ascending hierarchical classification, cladistic approach) were applied to the morphological characters of the adults of the genus Phlebotomus Rondani & Berté 1840. They confirm the reliability of the classic classifications, and also redefine the taxonomic and phylogenetic position of certain taxa. Thus, Spelaeophlebotomus Theodor 1948, Idiophlebotomus Quate & Fairchild 1961 and Australophlebotomus Theodor 1948 deserve generic rank. Among the vectors of leishmaniasis, the subgenus Phlebotomus Rondani & Berté 1840 is probably ancient. The results attribute an intermediate taxonomic and phylogenetic position to the taxa Euphlebotomus Theodor 1948 and Anaphlebotomus Theodor 1948, and reveal the probable artificial nature of the latter. The comparatively large numbers of species of subgenera Paraphlebotomus Theodor 1948, Synphlebotomus Theodor 1948 and, above all, Larroussius Nitzulescu 1931 and Adlerius Nitzulescu 1931, suggest that they are relatively recent. The development of adult morphological characters, the validity of their use in taxonomy and proposals for further studies are discussed.

Numerical taxonomy of Old World Phlebotominae (Diptera: Psychodidae). 2 - Restatement of classification upon subgeneric morphological characters

Numerical analyses (correspondence analyses, ascending hierarchical classification, and cladistics) were done with morphological characters of adult phlebotomine sandflies. The resulting classification largely confirms that of classical taxonomy for supra-specific groups from the Old World, though the positions of some groups are adjusted. The taxa Spelaeophlebotomus Theodor 1978, Idiophlebotomus Quate & Fairchild 1961, Australophlebotomus Theodor 1948 and Chinius Leng 1987 are notably distinct from other Old World groups, particularly from the genus Phlebotomus Rondani & Berté 1840. Spelaeomyia Theodor 1948 and, in particular, Parvidens Theodor & Mesghali 1964 are clearly separate from Sergentomyia França & Parrot 1920.

Flebotomíneos de Timóteo, estado de Minas Gerais, Brasil (Diptera: Psychodidae) / Sand flies in Timóteo, Minas Gerais, Brazil (Diptera: Psychodidae)

Casos esporácidos de leishmaniose tegumentar têm ocorrido no Município de Timóteo, Minas Gerais, basicamente na populaçäo rural. Para conhecer a fauna de flebotomíneos da regiäo, foram instaladas sete armadilhas luminosas de New Jersey na cidade, em sete diferentes bairros. As coletas foram realizadas no período de junho a outubro de 1994, dezembro de 1994 e janeiro a março de 1995, com um total de 3.240 horas por armadilha. Foram capturados 4.396 flebotomíneos, distribuídos em dois gêneros e vinte espécies: Brumptomyia cunhai, Brumptomyia nitzulescui, Lutzomyia (Nyssomyia) whitmani, Lutzomyia (Nyssomyia) intermedia, Lutzomyia quinquefer, Lutzomyia lenti, Lutzomyia (Pintomyia) fischeri, Lutzomyia migonei, Lutzomyia sallesi, Lutzomyia termitophila, Lutzomyia aragaoi, Lutzomyia borgmeieri, Lutzomyia (Psathyromyia) lutziana, Lutzomyia (Sciopemyia) sordelli, Lutzomyia (Pintomyia) pessoai, Lutzomyia (Trichopygomyia) longispina, Lutzomyia misionensis, Lutzomyia (Psychodopygus) davisi, Lutzomyia lanei, Lutzomyia (Pressatia) sp. A espécie L. (N.) whitmani foi a mais freqüente com 52,12 por cento, seguida de L. (N.) intermedia com 34,10 por cento, e ambas podem estar participando da transmissäo de leishmaniose cutânea na regiäo.

Report on a collection of Lutzomyia sand flies (Diptera: Psychodidae) from the Middle Solimoes (Amazonas, Brazil)

Fifty-two species of Lutzomyia sand flies were identified in contemporaneous samples totalling only 1875 individuals, collected at the same site in tall primary terra-firme rainforest, near the south bank of the Solimoes River. The most abundant species belonged to the subgenera Trichophoromyia and Nyssomyia. The subgenera Psathyromyia, Nyssomyia and Psychodopygus represented the greatest number of species. A new, aberrant species of the subgenus Psathyromyia (L. cutellata) and the female of Lu. souzacastroi are described. The Phlebotominae are proposed as a suitable indicator group for biogeographic and diversity studies.

[Isoenzymatic studies of Mediterranean populations of sandflies of the subgenus Larroussius]. / Etude isoenzymatique de populations Mediterraneennes de phlebotomes du sous-genre Larroussius.

Isoenzymes of hexokinase, phosphoglucomutase and glucosephosphate isomerase were examined, using agarose gel isoelectrofocusing, in populations of Phlebotomus ariasi (France), P. neglectus (Greece), P. perfilewi (Italy & Greece) and P. tobbi (Greece). Comparison between enzymatic profiles by numerical analysis separated Larroussius from Phlebotomus and Paraphlebotomus subgenera and brought together P. ariasi and P. neglectus. Nei genetic coefficients indicated similarities between populations of P. perfiliewi from Corfu and central Italy and between Corfuan and Cretan P. neglectus samples.

Phlebotomes of Paraguay species identification in 3 endemic areas: diptera, psychodydae, and phlebotominae

In an attemp to contribute to a better knowledge of the leishmania-transmitting insect species in endemic areas of Paraguay, phlebotomes were collected by night catch. In this way, species of insects could be identified and natural infections could also be discovered. Six-hundred and six females bugs were examined, from among which the following species were identified: Lutsomia migonei (11%), L. shannoni (13.6%, L. intermedia (20.3%), L. wakeri (0.2%), L. whitmani (60.4), L. fisheri (2.6%), L. longispinosa (0.7%), and L. cortelezzi (0.2%). The last three species were first described in Paraguay. L. whitmani was the most frequently found species. Insect catch was performed in four Paraguayan localities in three endemic areas, using Shannon light traps and human bait, Leishmania promastigotes were found in the Malpighi tubes of a specimen of L. whitmani. All the other species mentioned above are also considered as vectors for Leishmania. Very little study has been performed on Phlebotomines in Paraguay. In 1955, González y Arce Queirolo collected 5401 specimens in endemic areas, Nad identified the following species: Ph. whitmani, Ph. migonei, Ph. intermedius, Ph. monticulosus, dnd Ph. pessoai (3). These species were cited by Barreto (1950) in his report on the distribution of phlebotomines in America (1). Between 1984 and 1985, A. de Arias performed an epidemiological survey in Tavapy (Alto Paraná), collecting the following species: L. intermedie, L. migonei and L. whitmani (6). Although all the species quoted by the different authors are considered vectors for Leishmania, they failed to find specimens infected with the parasite. In the present work we report some data on the species found in three endemic zones as well as the method of catch employed, and finally we mention some aspects of the natural infection

La fauna flebotómica (Diptera Psychodidae) en Venezuela: I.Taxonomía y distribución geográfica / The phlebotomine fauna (Diptera Psychodidae) in Venezuela: I.Taxonomy and geographical distribution

Se presenta una lista actualizada de los Flebótomos de Venezuela 91 especies pertenecen al género Lutzomyia y 4 especies se situan en el género Brumptomyia. Treinta especies son consideradas antropofilicas y 28 tienen su localidad tipo en el País. Para cada una se refiere la distribución geográfica por Estado, proporcionando también los rangos altitudinales conocidos