Online catalogue of the Coleção de Flebotomíneos (FIOCRUZ/COLFLEB), a biological collection of American sand flies (Diptera: Psychodidae, Phlebotominae) held at Fiocruz Minas, Brazil.

The Coleção de Flebotomíneos ("Phlebotomine Collection"; FIOCRUZ/COLFLEB) held at Fiocruz Minas is a curated biological collection comprising approximately 80 000 individual specimens of 370 species of sand flies (Diptera: Psychodidae, Phlebotominae). These were mostly collected from the Americas in the last 80 years by entomologists interested in understanding and controlling the vector-borne disease leishmaniases. Since 2010, the metadata of each biological specimen held in FIOCRUZ/COLFLEB, including the back catalogue of those deposited in previous decades, has been digitized. Here, we present the resulting electronic catalog containing records for 72,624 of the specimens, including all of the available provenance information associated with each specimen. The catalog is published online through the speciesLink network and the Sistema de Informação sobre a Biodiversidade Brasileira (SiBBr).

Level of Knowledge and Risk Factors for Visceral Leishmaniasis in a Mining Area of Minas Gerais State, Brazil.

Aiming to optimize and adjust leishmaniasis prevention and control measures for the resident population of Pains, state of Minas Gerais, Brazil, a structured questionnaire containing conceptual questions and questions about household characteristics was used to evaluate knowledge level and exposure risk. A total of 396 individuals were interviewed revealing unscientific and fragmented knowledge about the subject for most of the studied population. The female population was found to have 1.68 times more chance of knowing about the disease than the male population, while highly educated individuals were found to have 2.92 times more chances of knowing about leishmaniasis compared to basic educated individuals. All of the respondents reported the presence of, at least, one risk factor, while ages ≥40 years were considered a protective factor compared to younger ages, indicating that older individuals are more likely to recognize risks and protect themselves against leishmaniasis. These results will contribute to the production of didactic materials for the population with respect to their previous knowledge and will provide a basis for control and prophylactic measures.

Tripartite interactions: Leishmania, microbiota and Lutzomyia longipalpis.

The microbial consortium associated with sandflies has gained relevance, with its composition shifting throughout distinct developmental stages, being strongly influenced by the surroundings and food sources. The bacterial components of the microbiota can interfere with Leishmania development inside the sandfly vector. Microbiota diversity and host-microbiota-pathogen interactions regarding New World sandfly species have yet to be thoroughly studied, particularly in Lutzomyia longipalpis, the primary vector of visceral leishmaniasis in Brazil.The native microbiota of different developmental stages and physiological conditions of Lu. longipalpis (Lapinha Cave), was described by culturing and 16s rRNA gene sequencing. The 16s rRNA sequencing of culture-dependent revealed 13 distinct bacterial genera (Bacillus, Enterococcus, Erwinia, Enterobacter, Escherichia, Klebsiella, Lysinibacillus, Pseudocitrobacter, Providencia, Pseudomonas, Serratia, Staphylococcus and Solibacillus). The in vitro and in vivo effects of each one of the 13 native bacteria from the Lu. longipalpis were analyzed by co-cultivation with promastigotes of L.i. chagasi, L. major, L. amazonensis, and L. braziliensis. After 24 h of co-cultivation, a growth reduction observed in all parasite species. When the parasites were co-cultivated with Lysinibacillus, all parasites of L. infantum chagasi and L. amazonensis died within 24 hours. In the in vivo co-infection of L.chagasi, L. major and L. amazonensis with the genera Lysinibacillus, Pseudocitrobacter and Serratia it was possible to observe a significant difference between the groups co-infected with the bacterial genera and the control group.These findings suggest that symbiont bacteria (Lysinibacillus, Serratia, and Pseudocitrobacter) are potential candidates for paratransgenic or biological control. Further studies are needed to identify the nature of the effector molecules involved in reducing the vector competence for Leishmania.

The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major.

For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization. Our attempts to exploit antibiotic treatment of colonized Phlebotomus duboscqi sand flies in order to improve their vector competency for Leishmania major resulted instead in flies that were refractory to the development of transmissible infections due to the inability of the parasite to survive and to colonize the anterior midgut with infective, metacyclic stage promastigotes. The parasite survival and development defect could be overcome by feeding the flies on different symbiont bacteria but not by feeding them on bacterial supernatants or replete medium. The inhibitory effect of the dysbiosis was moderated by lowering the concentration of sucrose (<30% w/v) used in the sugar feeds to maintain the colony. Exposure of promastigotes to 30% sucrose was lethal to the parasite in vitro. Confocal imaging revealed that the killing in vivo was confined to promastigotes that had migrated to the anterior plug region, corresponding to the highest concentrations of sucrose. The data suggest that sucrose utilization by the microbiota is essential to promote the appropriate osmotic conditions required for the survival of infective stage promastigotes in vivo.

Bacterial diversity of the American sand fly Lutzomyia intermedia using high-throughput metagenomic sequencing.

BACKGROUND: Parasites of the genus Leishmania cause a broad spectrum of diseases, collectively known as leishmaniasis, in humans worldwide. American cutaneous leishmaniasis is a neglected disease transmitted by sand fly vectors including Lutzomyia intermedia, a proven vector. The female sand fly can acquire or deliver Leishmania spp. parasites while feeding on a blood meal, which is required for nutrition, egg development and survival. The microbiota composition and abundance varies by food source, life stages and physiological conditions. The sand fly microbiota can affect parasite life-cycle in the vector. METHODS: We performed a metagenomic analysis for microbiota composition and abundance in Lu. intermedia, from an endemic area in Brazil. The adult insects were collected using CDC light traps, morphologically identified, carefully sterilized, dissected under a microscope and the females separated into groups according to their physiological condition: (i) absence of blood meal (unfed = UN); (ii) presence of blood meal (blood-fed = BF); and (iii) presence of developed ovaries (gravid = GR). Then, they were processed for metagenomics with Illumina Hiseq Sequencing in order to be sequence analyzed and to obtain the taxonomic profiles of the microbiota. RESULTS: Bacterial metagenomic analysis revealed differences in microbiota composition based upon the distinct physiological stages of the adult insect. Sequence identification revealed two phyla (Proteobacteria and Actinobacteria), 11 families and 15 genera; 87 % of the bacteria were Gram-negative, while only one family and two genera were identified as Gram-positive. The genera Ochrobactrum, Bradyrhizobium and Pseudomonas were found across all of the groups. CONCLUSIONS: The metagenomic analysis revealed that the microbiota of the Lu. intermedia female sand flies are distinct under specific physiological conditions and consist of 15 bacterial genera. The Ochrobactrum, Bradyrhizobium and Pseudomonas were the common genera. Our results detailing the constituents of Lu. intermedia native microbiota contribute to the knowledge regarding the bacterial community in an important sand fly vector and allow for further studies to better understand how the microbiota interacts with vectors of human parasites and to develop tools for biological control.

O papel da microbiota intestinal na competência vetorialdo Lutzomyia longipalpis para a Leishmania (Leishmania)infantum chagasi e a transmissão do parasito ao vertebradopela da picada / The role of the intestinal microbiota in vector competence Lutzomyia longipalpis for the Leishmania (Leishmania) infantum chagasi and transmission of the parasite to the vertebrate by the bite

As Leishmanioses são doenças causadas por protozoários do gênero Leishmania etransmitidas pela picada dos flebotomíneos. A doença apresenta diferentes manifestaçõesclínicas, como a leishmaniose visceral (LV). No Brasil a LV é causada pela subespécieLeishmania (L.) infantum chagasi e transmitida principalmente pelo vetor Lutzomyialongipalpis e nos últimos dez anos, a média anual de novos casos foi de 3.379. Oflebotomíneo, durante o repasto sanguíneo, pode ingerir o parasito que é direcionado aointestino médio do inseto, onde entra em contato com a microbiota. In vitro a ação dessamicrobiota sobre o parasito foi avaliada demonstrando a lise na parede celular de Leishmania(L.)infantum chagasi e Leishmania (V.) braziliensis causada por Serratia marcescens, porémainda não esta claro o seu papel no vetor. Neste trabalho foi feita a identificação porsequenciamento de DNA de isolados bacterianos do intestino médio de L. longipalpis de duaslocalidades brasileiras. Nas fêmeas de flebotomíeneos da Gruta da Lapinha foi encontradapela primeira vez a bactéria da família Enterobactereaceae, Providencia sp. Nas fêmeas deJacobina a bactéria encontrada foi a espécie Acinetobacter soli. As duas localidadesapresentaram maior número de bactérias do tipo bacilos Gram negativos. Os machos deJacobina não apresentaram crescimento bacteriano, diferentemente dos machos da Gruta daLapinha. Para análise da influência das bactérias no desenvolvimento do parasito, Fêmeas deL. longipalpis foram infectadas com Leishmania (L.) infantum chagasi e tratadas com doisantibióticos de ação principal sobre bactérias Gram negativas, carbenicilina e gentamicina. Osparasitos do grupo tratado com carbenicilina, além de serem capazes de se estabelecer noflebotomíneos após a digestão, tiveram um aumento no seu número. O grupo tratado comgentamicina apresentou uma queda já no segundo dia pós-infecção, tendo um aumento noquinto dia. Ao incubar as bactérias identificadas no intestino médio com L. (L.) infantumchagasi in vitro, foi possível ver que as bactérias interferem no crescimento do parasito.Analisamos também a expressão de peptídeos antimicrobianos. Gambicina e defensinaapresentaram diferença de expressão entre os grupos Não alimentados e Pós Digestão, sendoque a expressão foi maior em Não alimentados. No processo de interação, estabelecemos nolaboratório a transmissão de L.(L.) infantum chagasi diretamente via picada do flebotomíneo ahamsters e camundongos. Os dois modelos apresentaram uma variação de 4 a 15 mil parasitosinoculados na orelha dos vertebrados.

O papel da microbiota intestinal na competência vetorialdo Lutzomyia longipalpis para a Leishmania (Leishmania)infantum chagasi e a transmissão do parasito ao vertebradopela da picada

As Leishmanioses são doenças causadas por protozoários do gênero Leishmania etransmitidas pela picada dos flebotomíneos. A doença apresenta diferentes manifestaçõesclínicas, como a leishmaniose visceral (LV). No Brasil a LV é causada pela subespécieLeishmania (L.) infantum chagasi e transmitida principalmente pelo vetor Lutzomyialongipalpis e nos últimos dez anos, a média anual de novos casos foi de 3.379. Oflebotomíneo, durante o repasto sanguíneo, pode ingerir o parasito que é direcionado aointestino médio do inseto, onde entra em contato com a microbiota. In vitro a ação dessamicrobiota sobre o parasito foi avaliada demonstrando a lise na parede celular de Leishmania(L.)infantum chagasi e Leishmania (V.) braziliensis causada por Serratia marcescens, porémainda não esta claro o seu papel no vetor. Neste trabalho foi feita a identificação porsequenciamento de DNA de isolados bacterianos do intestino médio de L. longipalpis de duaslocalidades brasileiras. Nas fêmeas de flebotomíeneos da Gruta da Lapinha foi encontradapela primeira vez a bactéria da família Enterobactereaceae, Providencia sp. Nas fêmeas deJacobina a bactéria encontrada foi a espécie Acinetobacter soli. As duas localidadesapresentaram maior número de bactérias do tipo bacilos Gram negativos

Os machos deJacobina não apresentaram crescimento bacteriano, diferentemente dos machos da Gruta daLapinha. Para análise da influência das bactérias no desenvolvimento do parasito, Fêmeas deL. longipalpis foram infectadas com Leishmania (L.) infantum chagasi e tratadas com doisantibióticos de ação principal sobre bactérias Gram negativas, carbenicilina e gentamicina. Osparasitos do grupo tratado com carbenicilina, além de serem capazes de se estabelecer noflebotomíneos após a digestão, tiveram um aumento no seu número. O grupo tratado comgentamicina apresentou uma queda já no segundo dia pós-infecção, tendo um aumento noquinto dia. Ao incubar as bactérias identificadas no intestino médio com L. (L.) infantumchagasi in vitro, foi possível ver que as bactérias interferem no crescimento do parasito.Analisamos também a expressão de peptídeos antimicrobianos. Gambicina e defensinaapresentaram diferença de expressão entre os grupos Não alimentados e Pós Digestão, sendoque a expressão foi maior em Não alimentados. No processo de interação, estabelecemos nolaboratório a transmissão de L.(L.) infantum chagasi diretamente via picada do flebotomíneo ahamsters e camundongos. Os dois modelos apresentaram uma variação de 4 a 15 mil parasitosinoculados na orelha dos vertebrados