Identification of Lutzomyia longipalpis' using MALDI-TOF peptide/protein profiles.

Sand flies are vectors of great public health importance, since they constitute a group of hematophagous insects responsible for etiological agents transmission of zoonotic diseases such a visceral leishmaniasis. In face of the expansion of these diseases, efficient control strategies are needed which depend on comprehending the sand fly eco-epidemiology. In this regard, MALDI-TOF mass spectrometry has been used for bacteria, fungi and yeast detection studies through peptide/protein profiles. However, little is known about interference of biological factors associated with vector ecology, such as blood meal preferences and even sand fly age on the peptide/protein profiles. Thus, the present study aimed to evaluate the differences in peptide/protein profiles of the sand fly Lutzomyia longipalpis, by means of MALDI-TOF, due to the sand fly's age, sex, blood meal source and Leishmania infantum infection. Sample preparation was made removing both head and last abdomen segments keeping the thorax, its appendices and the rest of the abdomen. Five specimens per pool were used to obtain peptide/protein extract of which 1 µL solution was deposited over 1 µL MALDI matrix dried. Characteristic spectra were analyzed using principal coordinate analysis as well as indicator species analysis to discriminate differences in sand flies's peptide/protein profile by sex, age, blood meal source and L. infantum infection. The results show that the evaluated variables produced distinct peptide/protein profiles, demonstrated by the identification of specific diagnostic ions. It was found that the interference of biological factors should be taken into account when using the MALDI-TOF analysis of sand fly species identification and eco-epidemiological applications in field studies. Based on our results, we believe that it is possible to identify infected specimens and the source of blood meal in a collection of wild sand flies, serving to measure infectivity and understand the dynamics of the vector's transmission chain. Our results may be useful for epidemiological studies that look at the ecology of sand flies and leishmaniasis, as well as for raising awareness of biological characteristics' impact on peptide/protein profiles in sand fly species identification.

Toxicity of Trixis vauthieri DC essential oil on Lutzomyia longipalpis (Diptera, Psychodidae, Phlebotominae), a proven vector of visceral leishmaniasis in Brazil.

BACKGROUND OBJECTIVES: In Brazil, one of the visceral leishmaniasis control measures in urban environments is the elimination of Lutzomyia longipalpis, which occurs through the application of pyrethroid insecticides with residual action in homes and outbuildings. Due to the loss of sensitivity of this vector to these insecticides, the search for more efficient insecticide compounds against L. longipalpis has been intensified. The objective of this work was to evaluate the toxicity of Trixis vauthieri essential oil on adult sandflies of the species L. longipalpis, and identify the phytochemical composition of these essential oils. METHODS: Essential oils from leaves collected from T. vauthieri at different times were obtained at concentrations of 5, 10 and 20 mg/mL. Twenty sandflies were exposed to the essential oils and the mortality was evaluated after 1, 2, 4, 16, 24, 48 and 72 h. The chemical constituents of the essential oil were also identified. RESULTS: The essential oils of T. vauthieri at a concentration of 20 mg/mL were the most toxic to sandflies, reaching a mortality rate of 98.33% and 95%, respectively, after 72 h of exposure. The analysis of chemical constituents revealed the presence of triterpenes and/or steroids, tannins, flavonoids, alkaloids, saponins and coumarins. INTERPRETATION CONCLUSION: The results obtained suggest that T. vauthieri essential oil is fairly promising as an insecticidal potential against L. longipalpis. A more detailed analysis of the oil's phytochemical composition is necessary to identify active and pure compounds that can be used in vector control of visceral leishmaniasis.

Interactions between Leishmania parasite and sandfly: a review.

Leishmaniasis transmission cycles are maintained and sustained in nature by the complex crosstalk of the Leishmania parasite, sandfly vector, and the mammalian hosts (human, as well as zoonotic reservoirs). Regardless of the vast research on human host-parasite interaction, there persists a substantial knowledge gap on the parasite's development and modulation in the vector component. This review focuses on some of the intriguing aspects of the Leishmania-sandfly interface, beginning with the uptake of the intracellular amastigotes from an infected host to the development of the parasite within the sandfly's alimentary canal, followed by the transmission of infective metacyclic stages to another potential host. Upon ingestion of the parasite, the sandfly hosts an intricate repertoire of immune barriers, either to evade the parasite or to ensure its homeostatic coexistence with the vector gut microbiome. Sandfly salivary polypeptides and Leishmania exosomes are co-egested with the parasite inoculum during the infected vector bite. This has been attributed to the modulation of the parasite infection and subsequent clinical manifestation in the host. While human host-based studies strive to develop effective therapeutics, a greater understanding of the vector-parasite-microbiome and human host interactions could help us to identify the targets and to develop strategies for effectively preventing the transmission of leishmaniasis.

Molecular identification of Phlebotomus kandelakii apyrase and assessment of the immunogenicity of its recombinant protein in BALB/c mice.

Sand fly salivary proteins have immunomodulatory and anti-inflammatory features; hence, they are proven to perform important roles in the early establishment of Leishmania parasite in the vertebrate host. Among them, salivary apyrase with anti-hemostatic properties has a crucial role during the blood meal process. In the present study, a Genome-Walking method was used to characterize a full-length nucleotide sequence of Phlebotomus (P.) kandelakii apyrase (Pkapy). Bioinformatics analyses revealed that Pkapy is a ~ 36 kDa stable and hydrophilic protein that belongs to the Cimex family of apyrases. Moreover, recombinant proteins of Pkapy and P. papatasi apyrase (Ppapy) were over-expressed in Escherichia coli BL2 (DE3) and their antigenicity in BALB/c mice was evaluated. Dot-blot and ELISA results indicated that both recombinant apyrases could induce antibodies in BALB/c. Moreover, a partial cross-reactivity between Pkapy and Ppapy was found. In vitro stimulation of splenocytes from immunized mice with the recombinant proteins indicated cross-reactive T cell proliferative responses. Cytokine analysis revealed significant production of IFN-γ (p < 0.001) and IL-10 (p < 0.01) in response to Pkapy. In conclusion, the full-length nucleotide sequence and molecular characteristics of Pkapy were identified for the first time. Immunologic analyses indicated that Pkapy and Ppapy are immunogenic in BALB/c mice and show partial cross-reactive responses. The immunity to Pkapy was found to be a Th1-dominant response that highlights its potential as a component for an anti-Leishmania vaccine.

Evaluation of expression variations in virulence-related genes of Leishmania major after several culture passages compared with Phlebotomus papatasi isolated promastigotes.

Cutaneous leishmaniasis (CL) is a prevalent infectious disease with considerable morbidity annually. Here, we aimed to investigate the likely variations in gene expression of glycoprotein63 (gp63), heat shock protein 70 (HSP70), histone, arginase, cysteine protease B (CPB), Leishmania homologue of receptors for activated C kinase (LACK), small hydrophilic endoplasmic reticulum-associated protein (SHERP) in metacyclic promastigotes of L. major isolated from Phlebotomus papatasi sand flies and promastigotes excessively cultured in culture medium. The parasites were collected from suspected CL cases in Pasteur Institute of Iran, cultured and inoculated into the female BALB/c mice (2×106 promastigotes). Sand flies were trapped in Qom province, fed with the blood of euthanized infected mice and subsequently dissected in order to isolate the midgut including stomodeal valve. The metacyclic promastigotes were isolated from Ph. papatasi (Pro-Ppap) using peanut agglutinin test (PNA), then continuously cultured in RPMI-1640 medium enriched with fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) to reach stationary phase (Pro-Stat). The gene expression was evaluated in both parasitic stages (Pro-Ppap and Pro-Stat) using qRT-PCR. Out results showed a significant increased gene expression at Pro-Ppap stage for gp63 (P = 0.002), SHERP (P = 0.001) and histone (P = 0.026) genes, in comparison with Pro-Stat stage. Noticeably, significant changes were, also, demonstrated in 10th to 15th passages [gp63 (P = 0.041), arginase (P = 0.016), LACK (P = 0.025)] and in 5th to 20th passage (SHERP) (P = 0.029). In conclusion, the findings of the present study seem to be essential in designing Leishmania studies, in particular regarding host-parasite interaction, immunization and infectivity studies.

Chave fotográfica auxiliar na identificação de espécies de flebotomíneos (Diptera: Psychodidae: Phlebotominae) da Região Metropolitana da Grande São Paulo / Photographic key to aid in the identification of sand fly species (Diptera: Psychodidae: Phlebotominae) from the Greater São Paulo Metropolitan Region

Introdução: As leishmanioses são antropozoonoses que têm como agentes mais de 20 espécies do gênero Leishmania, transmitidas aos humanos através do repasto sanguíneo realizado por fêmeas de flebotomíneos (Diptera: Psychodidae: Phlebotominae) infectadas por esses protozoários. A elaboração de uma chave fotográfica para identificação de espécies de flebotomíneos para uso simultâneo com a chave dicotômica de Galati, 2018 pode auxiliar profissionais e leigos na identificação desses insetos, colaborando com estudos epidemiológicos sobre as leishmanioses e vigilância de seus vetores, bem como profilaxia e controle dessas doenças na população. Objetivo: Confeccionar chaves fotográficas auxiliares para a identificação de espécies de flebotomíneos com registros para a Grande São Paulo. Material e Métodos: Após levantamento das espécies que ocorrem na Grande São Paulo, foram fotografadas estruturas morfológicas de espécimes machos e fêmeas adultos, montados em lâminas pertencentes à coleção de referência da Faculdade de Saúde Pública da Universidade de São Paulo (FSP-USP) e Coleção didática do Laboratório de Entomologia em Saúde Pública da FSP-USP (LESP-Phlebotominae da FSP-USP), e também da Coleção de Fauna Sinantrópica do Município de São Paulo do Laboratório de Pesquisa em Fauna Sinantrópica (CFS do LABFAUNA). Para execução e tratamento das fotografias utilizou-se o microscópio óptico Axio Zeiss Lab. A1 acoplado a uma câmera fotográfica ligada a um computador com software Zeiss Zen 2012 (Blue edition). Resultados: Dos 39 municípios que compõem a região metropolitana da Grande São Paulo, em 32 deles, existem registros da ocorrência de flebotomíneos. Ao todo foram identificadas 41 espécies pertencentes às subtribos Brumptomyiina, Lutzomyiina, Psychodopygina e Sergentomyiina. Foram confeccionadas nove chaves fotográficas, sendo uma chave para ambos os sexos para identificação das subtribos a que pertencem, quatro chaves para identificação dos machos e quatro para identificação das fêmeas das respectivas subtribos. Foram produzidas aproximadamente 220 fotos de estruturas pertencentes à cabeça, tórax, abdômen e genitálias de machos e fêmeas. Conclusão: Os municípios com as maiores riquezas de espécies foram: São Paulo, Pirapora do Bom Jesus e ltapevi; e as menores, Poá e Ferraz de Vasconcelos. Pintomyia fischeri foi a espécie com registro em um maior número de municípios, seguida por Migonemyia migonei e Psychodopygus lloydi. Distinguiu-se pela primeira vez as fêmeas de Brumptomyia ortizi e Brumptomyia cardosoi, observando diferenças na comparação do tamanho do primeiro flagelômero em relação ao tamanho do labro-epifaringe.

lntroduction: Leishmaniases are anthropozoonoses whose agents are more than 20 species of the genus Leishmania, transmitted to humans through the blood meal performed by female sandflies (Diptera: Psychodidae: Phlebotominae) infected by these protozoa. The elaboration of a photographic key for identlfication of sand fly species for simultaneous use with the dichotomous key of Galati, 2018 can help professionals and lay people in the identification of these insects, collaborating with epidemiological studies on leishmaniases and surveillance of their vectors, as well as prophylaxis and control of these diseases in the population. Objective: To make auxiliary photographic keys to identify sand fly species with records for Greater São Paulo. Material and Methods: After surveying the species that occur in Greater São Paulo, morphological structures of their males and female adults mounted on slides were photographed. These specimens belonging to the reference collection of the Faculty of Public Health of the University of São Paulo (FSP-USP) and didactic collection from the Laboratory of Entomology in Public Health at FSP-USP (LESP-Phlebotominae at FSP-USP), and also from the Synanthropic Fauna Collection of the Municipality of São Paulo from the Research Laboratory of Synanthropic Fauna (CFS - LABFAUNA). For the execution and treatment of the photographs, the optical microscope Axio Zeiss Lab A1 was used. attached to a camera connected to a computer with Zeiss Zen 2012 software (Blue edition). Results: Of the 39 municipalities that make up the metropolitan region of Greater São Paulo, 32 of them have records of the occurrence of sand flies. A total of 41 species belonging to the Brumptomyiina, Lutzomyiina, Psychodopygina and Sergentomyiina subtribes were identified. Nine photographic keys were made, one key for both sexes to identify the subtribes to which they belong, four keys for males and four for females to identify the species of the respective subtribes. Approximately 220 photos of structures belonging to the head, thorax, abdomen and genitalia of males and females were taken. Conclusion: The municipalities with the highest species richness were São Paulo, Pirapora do Bom Jesus and ltapevi; and the smallest, Poá and Ferraz de Vasconcelos. Pintomyia físcheri was the species registered in a greater number of municipalities, followed by Migonemyia migonei and Psychodopygus lloydi. The females of Brumptomyia ortizi and Brumptomyia cardosoi were distinguished for the first time, observing differences in the comparison of the size of the first flagellomere in relation to the size of the labro-epipharynx.

Self-Hybridization in Leishmania major.

Genetic exchange between different Leishmania strains in the sand fly vector has been experimentally demonstrated and is supported by population genetic studies. In nature, opportunities for Leishmania interstrain mating are restricted to flies biting multiply infected hosts or through multiple bites of different hosts. In contrast, self-mating could occur in any infected sand fly. By crossing two recombinant lines derived from the same Leishmania major strain, each expressing a different drug-resistance marker, self-hybridization in L. major was confirmed in a natural sand fly vector, Phlebotomus duboscqi, and in frequencies comparable to interstrain crosses. We provide the first high resolution, whole-genome sequencing analysis of large numbers of selfing progeny, their parents, and parental subclones. Genetic exchange consistent with classical meiosis is supported by the biallelic inheritance of the rare homozygous single nucleotide polymorphisms (SNPs) that arose by mutation during the generation of the parental clones. In contrast, heterozygous SNPs largely failed to be transmitted in Mendelian ratios for reasons not understood. SNPs that were heterozygous in both parents, however, recombined to produce homozygous alleles in some hybrids. For trisomic chromosomes present in both parents, transmittal to the progeny was only altered by self-hybridization, involving a gain or loss of somy in frequencies predicted by a meiotic process. Whole-genome polyploidization was also observed in the selfing progeny. Thus, self-hybridization in Leishmania, with its potential to occur in any infected sand fly, may be an important source of karyotype variation, loss of heterozygosity, and functional diversity. IMPORTANCE Leishmania are parasitic protozoa that cause a wide spectrum of diseases collectively known as the leishmaniases. Sexual reproduction in Leishmania has been proposed as an important source of genetic diversity and has been formally demonstrated to occur inside the sand fly vector midgut. Nevertheless, in the wild, opportunities for genetic exchange between different Leishmania species or strains are restricted by the capacity of different Leishmania strains to colonize the same sand fly. In this work, we report the first high resolution, whole-genome sequence analysis of intraclonal genetic exchange as a type of self-mating in Leishmania. Our data reveal that self-hybridization can occur with comparable frequency as interstrain mating under experimental lab conditions, leading to important genomic alterations that can potentially take place within every naturally infected sand fly.

Study on the zoonotic cycle of tegumentary leishmaniasis in an endemic area of a metropolitan region in the Northeastern region of Brazil.

This study was conducted to characterize the transmission cycle of the tegumentary leishmaniasis (TL) in an old colonization area at Pernambuco State, Brazil. The aims were to identify autochthonous cases, sandflies fauna, domestic animals as possible reservoir hosts and the Leishmania species involved in this endemic area. A total of 168 suspected human cases of TL and 272 domestic animals (canine, feline, equine, goat, and sheep) were included. The sandflies were captured and identified by species. Patients were predominantly male and the average age was 37+18.1 years old. Of 85 patients who had skin lesions, 25.6% of them had direct positive smears for TL and 34 isolates were identified as Leishmania (Viannia) braziliensis. The confirmation for TL diagnosed by molecular detection (PCR) was almost three times more sensitive than the direct test [p < 0.001; PR = 2.72] associated with clinical examination. The Kappa test on PCR between two different specimens, biopsy, and skin lesion swab was 60.8% (p < 0.001). More than 200 specimens of sandflies (80 males and 159 females) were captured and identified as Lutzomyia whitmani (99.6%) and Lu. evandroi (0.4%). The detection of L. (V.) braziliensis by Real-Time PCR in the blood of a captured fed female was positive in 59.3% of Lu. whitmani. Of the 272 domestic animals included, 61.76% were male (n = 168). Thirty-six animals (13.2%) had lesions compatible with TL (34 dogs, 1 cat and 1 sheep) and 3 of them, all dogs, had lesions on the snout, showing destruction of cartilage and mucosa. The study suggests the participation of domestic animals as possible reservoirs. However, further studies are necessary to better understand the transmission cycle and take recommended measures in order to control the disease.

Isolation, characterization, and functional study of extracellular vesicles derived from Leishmania tarentolae.

Leishmania (L.) species are protozoan parasites with a complex life cycle consisting of a number of developmental forms that alternate between the sand fly vector and their host. The non-pathogenic species L. tarentolae is not able to induce an active infection in a human host. It has been observed that, in pathogenic species, extracellular vesicles (EVs) could exacerbate the infection. However, so far, there is no report on the identification, isolation, and characterization of L. tarentolae EVs. In this study, we have isolated and characterized EVs from L. tarentolae GFP+ (tEVs) along with L. major GFP+ as a reference and positive control. The EVs secreted by these two species demonstrated similar particle size distribution (approximately 200 nm) in scanning electron microscopy and nanoparticle tracking analysis. Moreover, the said EVs showed similar protein content, and GFP and GP63 proteins were detected in both using dot blot analysis. Furthermore, we could detect Leishmania-derived GP63 protein in THP-1 cells treated with tEVs. Interestingly, we observed a significant increase in the production of IFN-γ, TNF-α, and IL-1ß, while there were no significant differences in IL-6 levels in THP-1 cells treated with tEVs following an infection with L. major compared with another group of macrophages that were treated with L. major EVs prior to the infection. Another exciting observation of this study was a significant decrease in parasite load in tEV-treated Leishmania-infected macrophages. In addition, in comparison with another group of Leishmania-infected macrophages which was not exposed to any EVs, tEV managed to increase IFN-γ and decrease IL-6 and the parasite burden. In conclusion, we report for the first time that L. tarentolae can release EVs and provide evidence that tEVs are able to control the infection in human macrophages, making them a great potential platform for drug delivery, at least for parasitic infections.

Na+/K+-ATPase Activation by cAMP in the Midgut of Lutzomyia longipalpis (Lutz & Neiva, 1912; Diptera: Psychodidae).

Lutzomyia longipalpis (Lutz & Neiva, 1912) females have been intensively studied regarding the regulation of midgut pH. The mechanisms involved in pH regulation are complex, and some aspects remain to be clarified. Here, we investigated the role of the Na+/K+-ATPase pump as an electrochemical potential generator and its modulation by the second messenger cAMP in the midgut of female L. longipalpis. Our results suggest that not only may Na+/K+-ATPase be the main generator of an electrochemical potential across membranes in the midgut of female L. longipalpis, but also its activity is positively regulated by cAMP. cAMP-mediated Na+/K+-ATPase pump activity might be necessary to maintain the transport of the nutrients produced during blood digestion.

The knowns and unknowns of the efficacy of neem oil (Azadirachta indica) used as a preventative measure against Leishmania sand fly vectors (Phlebotomus genus).

Since domestic dogs are the main reservoir hosts of Leishmania infantum throughout the world, they are the main focus in terms of controlling zoonotic visceral leishmaniosis. To protect dogs from leishmaniosis, chemical repellents of durable efficacy are available in the form of collars, spot-on and sprays. However, the negative effects of chemical pesticides on the environment are well established as they affect animals and plants. This phenomenon has created the need for safer and more environmentally friendly substitutes. Plant extract-based insecticides and/or repellents have therefore been increasingly used by pet owners and veterinarians. Several botanical products have been tested as insecticides and/or repellents against a variety of bloodsucking arthropods that transmit human diseases. Among the products tested against Leishmania vectors, neem oil containing azadirachtin is the most studied. This study reviews the scientific literature concerning the efficacy of neem oil (azadirachtin-based products) against phlebotomine sand fly bites. A questionnaire was also administered to assess Italian veterinarians' attitudes to the use of neem oil. The survey was anonymous and consisted of three closed-ended questions. According to the data reported in the literature, the efficacy of neem oil in reducing the risk of sand fly bites has been tested against Phlebotomus papatasi, Phlebotomus perniciosus, Phlebotomus argentipes, Phlebotomus orientalis and Phlebotomus bergeroti. The efficacy of the products tested was expressed in percentages, ranging from 74.9% to 100%. The protection time was only available for six out of eight studies, ranging from "only during the first hour" (minimum protection time) to "all night" (expected maximum protection time). As regards the attitude to recommending the use of neem oil, 208 veterinarians participated in the online survey. Of the 126 veterinarians who recommended natural products, 119 (94.44%) reported that they recommended the use of neem oil-based products. Considering the limited data on the duration of protection and the dose of the active ingredient, more studies are required on the efficacy of neem oil-based products in reducing the risk of contracting canine leishmaniosis. These studies should also refer specifically to the concentration of the active ingredient as well as the interval of administration. Until such results are available, the use of azadirachtin-based products as the only topical products for the prevention of leishmaniosis in dogs is not recommended.

cAMP: A second messenger involved in the mechanism of midgut alkalinization in Lutzomyia longipalpis.

The sand fly Lutzomyia longipalpis is the main vector of Leishmania infantum in the Americas. Female sand flies ingest sugar-rich solutions and blood, which are digested in the midgut. Digestion of nutrients is an essential function performed by digestive enzymes, which require appropriate physiological conditions. One of the main aspects that influence enzymatic activity is the gut pH, which must be tightly controlled. Considering second messengers are frequently involved in the coordination of tightly regulated physiological events, we investigated if the second messenger cAMP would participate in the process of alkalinization in the abdominal midgut of female L. longipalpis. In midguts containing the indicator dye bromothymol-blue, cAMP stimulated the alkalinization of the midgut lumen. Through another technique based on the use of fluorescein as a pH indicator, we propose that cAMP is involved in the alkalinization of the midgut by activating HCO3- transport from the enterocyte's cytoplasm to the lumen. The results strongly suggested that the carrier responsible for this process would be a HCO3- /Cl- antiporter located in the enterocytes' apical membrane. Hematophagy promotes the release of alkalinizing hormones in the hemolymph; however, when the enzyme adenylyl cyclase, responsible for cAMP production, was inhibited, we observed that the hemolymph from blood-fed L. longipalpis' females did not stimulate midgut alkalinization. This result indicated that hormone-stimulated alkalinization is mediated by cAMP. In the present study, we provide evidences that cAMP has a key role in the control of intestinal pH.

Avaliação do efeito dos compostos biológicos Bacillus thuringiensis var. israelensis e Espinosade na sobrevida de larvas de Lutzomyia longipalpis (Diptera: Psychodidae) sob condições de laboratório / Evaluation of the effect of biological compounds Bacillus thuringiensis var. israelensis and Spinosade on the survival of Lutzomyia longipalpis (Diptera: Psychodidae) larvae under laboratory conditions

A leishmaniose visceral (LV) continua sendo um grave problema de Saúde Pública principalmente pela expansão a localidades urbanas do Brasil nas últimas décadas. Esta zoonose é causada pelo protozoário Leishmania infantum, transmitida principalmente pelo flebotomíneo Lutzomyia longipalpis e tem o cão doméstico como principal reservatório vertebrado. As formas de controle preconizadas no Brasil têm mostrado baixa efetividade na redução de casos e de óbitos, com dificuldades tanto no controle de reservatórios (dificuldades para execução da eutanásia) bem como no controle de vetores. Estes fatos trazem à tona a necessidade de estudos de formas alternativas que possam contribuir em um contexto de manejo integrado. O objetivo foi comparar a suscetibilidade e a sobrevida de formas imaturas de Lutzomyia longipalpis aos larvicidas biológicos Bacillus thuringiensis var. israelensis (Bti) e Espinosade. Foram realizados experimentos com doses de 10 mg/g e 20 mg/g dos compostos Bti e Espinosade aplicados no alimento oferecido aos frascos contendo larvas de Lu. longipalpis de terceiro estádio larval. Para cada concentração/composto, foram expostas 30 larvas em 3 réplicas. Como controle, foram utilizadas 30 larvas com aplicação de água. Nos tratamento com Bti foi observada mortalidade de 18,8% na concentração de 10 mg/g e de 50,4% na concentração de 20 mg/g. Quanto ao tempo de desenvolvimento larval e de pupas, observou-se que o tempo mediano de emergência dos alados foi 2,35 vezes maior na concentração de 20 mg/g e 1,9 vezes maior na concentração 10 mg/g, comparados ao grupo controle. Para o composto Espinosade nestas concentrações iniciais, a mortalidade foi de 100%. O ensaio foi repetido com o Espinosade em 4 diluições da menor dose testada anteriormente (10 mg/g), na proporção de 1:10, 1:20. 1:50 e 1:100, com resultados de mortalidade de 71%, 66,5%, 2,1% e 1,4% respectivamente. A DL90 foi estimada em 6,3 mg/g. Os resultados sugerem potencial uso dos dois compostos para o controle de imaturos de Lu. longipalpis, aplicado de forma diluída diretamente nos potenciais criadouros destes insetos, como forma complementar às ações de controle integrados em áreas de transmissão de LV.

Visceral leishmaniasis (LV) is a serious issue in public health in Brazil mainly by the spread in urban areas in the last decades. This zoonosis is caused by the protozoan Leishmania infantum, transmitted mainly by Lutzomyia longipalpis. The control measures recommended in Brazil showed low effectiveness in the reduction of cases, with difficulties in the control of domestic reservoirs as well as the vector control. These facts highlight the necessity of studies evaluating alternative methods that can contribute in an integrated management approach. The objective was to compare the susceptibility and the survival of Lutzomyia longipalpis immature forms to the biological compounds Bacillus thuringiensis var. israelensis and Spinosade. Experiments with doses of 10 mg/g and 20 mg/g of each compound, Bacillus thuringiensis var. israelensis and Spinosade were realized applying in Petri dishes containing larvae third instar. For each stage 30 larvae were exposed to each concentration compound and the observation was realized in triplicate. As control 30 larvae of each instar in which was applied water. The treatments with Bti resulted in mortality of 18.78% at a concentration of 10 mg/g and of 50.42% at a concentration of 20 mg/g and delay in the development of immature forms, with a median time for emergence of adults forms 2.35 times higher in the first group and 1.9 times higher in the second compared to the control group. For the Spinosad compound at these initial concentrations, the mortality was 100%. The assay was repeated with Espinosade in 4 dilutions of the lowest dose tested previously (10 mg/g), in the proportion of 1:10, 1:20. 1:50 and 1:100, with mortality results of 71%, 66.5%, 2.1% and 1.4% respectively. LC90 was estimated at 6.3 mg/g. The results suggest potential use of the two compounds for the control of Lu. Longipalpis immatures, applied in a diluted form directly to the potential breeding sites of these insects, as a complement to integrated control actions in areas of VL transmission.

The transmission of visceral leishmaniasis in the municipality of Guarujá, on the Coast of São Paulo state, Brazil

ABSTRACT OBJECTIVE To perform an entomological survey, evaluating the circulation of Leishmania spp. in sand flies captured from the new foci of visceral leishmaniasis (VL) in the coastal region of São Paulo state. METHODS Sand flies were captured from November 2016 to September 2018 using light traps of the Centers for Disease Control (CDC), in the neighborhood where VL cases were reported. Leishmania spp. circulation was evaluated by gut dissection and molecular analysis of the females captured. RESULTS Nyssomyia intermedia was the more frequent species (90,7%) within the 1,203 sand flies captured. We found no flagellates in dissected females, but two pools containing females of Ny. intermedia presented DNA of L. infantum. CONCLUSION Our results suggest that Ny. intermedia might be involved in the establishment of this new VL focus in Sao Paulo. However, before incriminating this species as a L. infantum vector, further studies should investigate other vectorial capacity parameters, including competence, survival, and feeding habits.

Levantamento da fauna e infecção por Leishmania spp em flebotomíneos ( Diptera: Psychodidae) na localidade de Catimbau Grande município de Rio Bonito estado do Rio de Janeiro / Fauna survey and infection by Leishmania spp in sandflies (Diptera: Psychodidae) in the locality of Catimbau Grande municipality of Rio Bonito state of Rio de Janeiro

As leishmanioses (tegumentar e visceral) são antropozoonoses causadas por protozoários do gênero Leishmania apontadas como doenças negligenciadas. São endêmicas em 98 países e consideradas neste contexto como um grande problema de saúde pública global. Fatores como desmatamento, migração e urbanização desordenada alteram os habitats dos flebotomíneos, além de provocar a mudança e distribuição de hospedeiros e reservatórios em seus nichos originais. O conhecimento da biologia dos flebotomíneos e o estudo da sua presença em determinada área é de extrema importância para elucidar a epidemiologia local da doença. O objetivo do estudo foi realizar um levantamento da fauna flebotomínica e o diagnóstico de infecção natural de fêmeas de flebotomíneos por Leishmania spp. A área de estudo foi Catimbau Grande, um povoado do município de Rio Bonito no Estado do Rio de Janeiro. Os insetos foram capturados com armadilhas luminosas HP (tipo CDC) instaladas aproximadamente a 1m do solo no peridomicílio e no extradomicílio, no período de janeiro a julho de 2019, as capturas foram realizadas a cada 15 dias em média. Foram registrados em fichas de campo dados como altitude, temperatura, umidade e fase lunar. No laboratório foi realizada a triagem dos insetos capturados e os flebotomíneos separados para montagem em lâmina e identificação. Foram identificados 157 flebotomíneos (67 fêmeas e 90 machos), duas espécies foram identificadas: Nyssomyia intermedia e Migonemyia migonei. Nenhuma das fêmeas analisadas para infecção natural apresentou positividade para Leishmania spp. (AU)

Taxonomia integrativa de espécies da série Shannoni de Psathyromyia (Psathyromyia) Barretto, 1962 (Diptera, Psychodidae, Phlebotominae) do estado de São Paulo / Integrative taxonomy of Psathyromyia (Psathyromyia) Barretto, 1962 Shannoni series species (Diptera, Psychodidae, Phlebotominae) from São Paulo state

Os flebotomíneos são dípteros que habitam ambientes florestais e antrópicos e são encontrados comumente em cavernas. Com exceção da Antártica, habitam todos os demais continentes. Atualmente o grupo é composto por pouco mais de 1.000 espécies. As fêmeas são hematófagas e várias espécies apresentam relevância em saúde pública como vetores de agentes de leishmanioses, arboviroses e bartonelose. A taxonomia dos flebotomíneos vem se desenvolvendo desde o início do século XX amplamente baseada em estudos morfológicos e morfométricos lineares das formas adultas. Atualmente tem sido recomendada a abordagem integrativa na delimitação das espécies, que além dos estudos clássicos, utiliza informações moleculares, das formas imaturas, geometria e ecologia, além de outros parâmetros. O objetivo deste projeto foi contribuir para um melhor conhecimento das quatro espécies do subgênero Psathyromyia que ocorrem no estado de São Paulo, duas das quais foram recentemente descritas (Pa. baratai e Pa. ribeirensis) e as duas outras revalidadas (Pa. bigeniculata e Pa. limai), a fim de encontrar diferenças entre as fêmeas dessas espécies, morfologicamente muito semelhantes. Coleções entomológicas foram consultadas, e para a coleta de espécimes foram amostrados ambientes florestais e antrópicos do estado de São Paulo e Mato Grosso do Sul, utilizando diferentes armadilhas de coleta. Foram realizadas análises discriminantes na morfometria linear de estruturas da cabeça e na morfometria geométrica das asas, além de análises moleculares do fragmento do gene citocromo c oxidase subunidade I (COI) por meio da construção de uma árvore de Neighbor-Joining (NJ) e método de delimitação de espécies ABGD (Automatic Barcode Gap Discovery), com a inclusão de sequências de Pa. abonnenci, Pa. bigeniculata e Pa. shannoni depositadas no GenBank. Como resultados, as análises discriminantes da morfometria linear possibilitaram a separação das espécies, exceto o par Pa. ribeirensis/Pa. limai. Para a morfometria geométrica, o tamanho do centroide evidenciou diferenças entre as asas de Pa. limai e Pa. ribeirensis em relação à de Pa. bigeniculata e a análise de variância de procrustes evidenciou a separação de todas as espécies, mesmo após a validação cruzada, exceto entre Pa. ribeirensis e Pa. limai, sugerindo que essas duas espécies são indistinguíveis morfometricamente. As análises do fragmento do gene COI foram úteis para separar as espécies. Psthyromyia bigeniculata apresentou altos valores de distância intraespecífica (4,7%), que pode representar diversidade críptica. De modo geral, a taxonomia integrativa foi útil para separar três das quatro espécies estudadas e corrobora a retirada de Pa. limai e Pa. bigeniculata da sinonímia de Pa. shannoni, bem como a descrição de Pa. baratai como um táxon diferente. Fêmeas de Pa. ribeirensis permanecem indistinguíveis daquelas de Pa. limai pela morfologia e morfometrias linear e geométrica.

Sand flies are dipterans that inhabit forest and anthropic environments and are commonly found in caves. Except for Antarctica, they inhabit all other continents. Currently the group comprises about 1,000 species. Females are hematophagous and several species are relevant for public health as leishmaniasis, arboviruses and bartonellosis agent vectors. The taxonomy of sand flies has been developing since the beginning of the 20th century, largely based on morphological and linear morphometric studies of the adult forms. Currently, an integrative approach to species delimitation has been recommended, which, in addition to classical studies, uses molecular information, immature forms, geometry and ecology, among other parameters. The aim of this project was to contribute to a better understanding of the four species of the Psathyromyia subgenus that occur in the state of São Paulo, two of them were recently described (Pa. baratai and Pa. ribeirensis) and the two others revalidated (Pa. bigeniculata and Pa. limai), in order to find differences between the females of the species, which have similar morphological traits. Museum entomological collections were consulted, and wild-caught specimens were collected in forest and anthropic environments in the states of São Paulo and Mato Grosso do Sul, using different collection traps. Discriminant analyzes were performed on the linear morphometry of head structures and on geometric morphometry of wing, in addition to molecular analyzes of the cytochrome c oxidase subunit I (COI) gene fragment by means of the construction of a Neighbor-Joining (NJ) tree and species delimitation method ABGD (Automatic Barcode Gap Discovery), wth the inclusion of Pa. abonnenci, Pa. bigeniculata and Pa. shannoni sequences deposited in GenBank. As a result, the discriminant analyzes of linear morphometry allowed the separation of species, except for the pair Pa. ribeirensis/Pa. limai. For the geometric morphometry, the centroid size showed differences between wings of Pa. limai and Pa. ribeirensis in relation to that of Pa. bigeniculata, while the procrustes analysis of variance showed the separation of all species even after cross-validation, except between Pa. ribeirensis and Pa. limai, suggesting that these two species are morphometrically indistinguishable. COI gene fragment analyzes were useful to separate the species. Psthyromyia bigeniculata showed high intraspecific distance values (4.7%), which may represent cryptic diversity. In general, the integrative taxonomy was useful to separate three of the four species studied and corroborates the resurrection of Pa. limai and Pa. bigeniculata from the synonymy of Pa. shannoni, as well as the description of Pa. baratai as a different taxon. Females of Pa. ribeirensis remain indistinguishable from those of Pa. limai for linear and geometric morphology and morphology.

Cutaneous leishmaniasis–Awareness, knowledge and practices among general population in rural and urban areas in Malakand region, Pakistan / Leishmaniose cutânea - conscientização, conhecimentos e práticas entre a população em geral nas áreas rurais e urbanas da região de Malakand, Paquistão

Malakand region is an endemic area for cutaneous leishmaniasis (CL). However, there are limited number of studies of this disease in Pakistan. Therefore, a study was conducted to understand the level of awareness attitude and practice among the residents of Makaland towards CL and the disease vectors. This study adopted a cross-sectional approach with a total of 400 respondents (n=93 rural and n= 307 urban). Overall, the population in Malakand region (61.2%) were well-informed in the role of sand fly in transmitting diseases, but most lack knowledge on the vector's behavior and almost a quarter (24.5%) were unable to provide knowledge on proper control measures. Alarmingly, the practice and attitudes of the general population was not satisfactory as close to half (49.8%) of the population did adopt any control method. This study calls for increase in awareness through health education campaign to reduce the risk of cutaneous leishmaniasis outbreaks in the future.

A região de Malakand é uma área endêmica para leishmaniose cutânea (CL). No entanto, há um número limitado de estudos sobre esta doença no Paquistão. Portanto, foi realizado um estudo para entender o nível de atitude e prática de conscientização entre os residentes de Makaland em relação aos CL e os vetores da doença. Este estudo adotou uma abordagem transversal com um total de 400 entrevistados (n = 93 rural e n = 307 urbano). No geral, a população da região de Malakand (61,2%) estava bem informada sobre o papel da mosca na transmissão de doenças, mas a maioria não possui conhecimento sobre o comportamento do vetor e quase um quarto (24,5%) foi incapaz de fornecer conhecimento sobre medidas de controle adequadas . De maneira alarmante, a prática e as atitudes da população em geral não foram satisfatórias, pois cerca da metade (49,8%) da população adotou algum método de controle. Este estudo apela ao aumento da conscientização por meio de campanhas de educação em saúde para reduzir o risco de surtos de leishmaniose cutânea no futuro.

Cutaneous leishmaniasis - Awareness, knowledge and practices among general population in rural and urban areas in Malakand region, Pakistan / Leishmaniose cutânea - conscientização, conhecimentos e práticas entre a população em geral nas áreas rurais e urbanas da região de Malakand, Paquistão

Malakand region is an endemic area for cutaneous leishmaniasis (CL). However, there are limited number of studies of this disease in Pakistan. Therefore, a study was conducted to understand the level of awareness attitude and practice among the residents of Makaland towards CL and the disease vectors. This study adopted a cross-sectional approach with a total of 400 respondents (n=93 rural and n= 307 urban). Overall, the population in Malakand region (61.2%) were well-informed in the role of sand fly in transmitting diseases, but most lack knowledge on the vector's behavior and almost a quarter (24.5%) were unable to provide knowledge on proper control measures. Alarmingly, the practice and attitudes of the general population was not satisfactory as close to half (49.8%) of the population did adopt any control method. This study calls for increase in awareness through health education campaign to reduce the risk of cutaneous leishmaniasis outbreaks in the future.

A região de Malakand é uma área endêmica para leishmaniose cutânea (CL). No entanto, há um número limitado de estudos sobre esta doença no Paquistão. Portanto, foi realizado um estudo para entender o nível de atitude e prática de conscientização entre os residentes de Makaland em relação aos CL e os vetores da doença. Este estudo adotou uma abordagem transversal com um total de 400 entrevistados (n = 93 rural e n = 307 urbano). No geral, a população da região de Malakand (61,2%) estava bem informada sobre o papel da mosca na transmissão de doenças, mas a maioria não possui conhecimento sobre o comportamento do vetor e quase um quarto (24,5%) foi incapaz de fornecer conhecimento sobre medidas de controle adequadas . De maneira alarmante, a prática e as atitudes da população em geral não foram satisfatórias, pois cerca da metade (49,8%) da população adotou algum método de controle. Este estudo apela ao aumento da conscientização por meio de campanhas de educação em saúde para reduzir o risco de surtos de leishmaniose cutânea no futuro.

Stage-Specific Class I Nucleases of Leishmania Play Important Roles in Parasite Infection and Survival.

Protozoans of the genus Leishmania are the causative agents of an important neglected tropical disease referred to as leishmaniasis. During their lifecycle, the parasites can colonize the alimentary tract of the sand fly vector and the parasitophorous vacuole of the mammalian host, differentiating into distinct stages. Motile promastigotes are found in the sand fly vector and are transmitted to the mammalian host during the insect blood meal. Once in the vertebrate host, the parasites differentiate into amastigotes and multiply inside macrophages. To successfully establish infection in mammalian hosts, Leishmania parasites exhibit various strategies to impair the microbicidal power of the host immune system. In this context, stage-specific class I nucleases play different and important roles related to parasite growth, survival and development. Promastigotes express 3'-nucleotidase/nuclease (3'-NT/NU), an ectoenzyme that can promote parasite escape from neutrophil extracellular traps (NET)-mediated death through extracellular DNA hydrolysis and increase Leishmania-macrophage interactions due to extracellular adenosine generation. Amastigotes express secreted nuclease activity during the course of human infection that may be involved in the purine salvage pathway and can mobilize extracellular nucleic acids available far from the parasite. Another nuclease expressed in amastigotes (P4/LmC1N) is located in the endoplasmic reticulum of the parasite and may be involved in mRNA stability and DNA repair. Homologs of this class I nuclease can induce protection against infection by eliciting a T helper 1-like immune response. These immunogenic properties render these nucleases good targets for the development of vaccines against leishmaniasis, mainly because amastigotes are the form responsible for the development and progression of the disease. The present review aims to present and discuss the roles played by different class I nucleases during the Leishmania lifecycle, especially regarding the establishment of mammalian host infection.

Leishmania infantum strains from cats are similar in biological properties to canine and human strains.

Zoonotic visceral leishmaniosis is a worldwide severe disease caused by Leishmania infantum, a protozoan that has phlebotomine sand flies as vectors and dogs as primary reservoir hosts. Over the last few decades, cats have been regarded as an indisputable piece within the ecological system in which L. infantum is maintained indefinitely. However, little is known about feline strains, including their phenotypic plasticity and infectivity. In this study, the phenotypic behaviour of seven L. infantum feline strains was compared to those of well-characterised counterparts isolated from two dogs and two humans in terms of growth profile, adaptive capacity under several stress conditions, susceptibility to antileishmanial drugs, and infectivity to host cells. Feline strains displayed a similar growth profile, survival capacity, and ability to infect feline, canine, and human monocyte-derived primary macrophages. Furthermore, multivariate cluster analysis suggested that most strains studied did not display distinctive phenotypic features. To our knowledge, this is the first study to analyse the phenotypic behaviour of feline L. infantum strains. This study brings new insights into the hypothetical role of cats as reservoir hosts of L. infantum since the parasites found in them are phenotypically identical to those of dogs and humans. However, further studies on the transmission dynamics should be encouraged to fully establish the status of cats in the maintenance of L. infantum foci.