Genetic variation of sand flies (Diptera: Psychodidae) in Gampaha and Kurunegala districts of Sri Lanka: Complementing the morphological identification

Objective: To identity the variation of sand flies in the Gampaha and Kurunegala districts of Sri Lanka and to assess DNA barcoding as a complementing method for morphological identification. Methods: A total of 38 441 sand flies were collected from selected localities in Gampaha and Kurunegala districts using standard entomological techniques from May 2017 to December 2018. Specimens were identified using morphological features and compared with mitochondrial cytochrome C oxidase subunit I gene- based DNA barcoding as an alternative tool. Results: Morphological and molecular identification confirmed the presence of four species under two genera (Phlebotomus and Sergentomyia). Phlebotomus argentipes was the predominant species, followed by Sergentomyia (S.) punjabensis, S. babu insularis, and an unidentified Sergentomyia sp. Phlebotomus argentipes showed a clear genetic differentiation from other species. S. babu insularis and S. punjabensis showed a higher genetic affinity to each other than the unidentified species. The unidentified Sergentomyia species is morphologically similar to S. zeylanica, but differs only in clavate gonostyle. Conclusions: DNA barcoding is an effective technique for the identification of sand flies. Further studies using molecular techniques will improve the knowledge of the cryptic diversity of Sri Lankan sand fly fauna. Establishing a reliable and standardized identification system for sand fly species in Sri Lanka is recommended.

Characterization of a municipality as free of canine visceral leishmaniasis in the context of One Health / Caracterização de um município como livre de leishmaniose visceral canina em um contexto de Saúde Única

Abstract Dogs are the main urban reservoir of Leishmania infantum, the causative agent of visceral leishmaniasis (VL), which is transmitted by sand flies. In the state of Paraná, the first detection of a positive dog for VL was in 2014, this year Paraná lost free status for this disease (VL). The objectives of this study were to determine the prevalence of canine visceral leishmaniasis in Palotina, the occurrence of vectors that may transmit Leishmania infantum, and the number of notifications of human visceral leishmaniasis cases from period 2010 to 2020. To determine the occurrence of canine visceral leishmaniasis, blood samples from 204 dogs were analyzed using the rapid test DPP® to detect anti-L. infantum antibodies. To investigate the occurrence of potential vectors, monthly collections were made at 18 points within the urban area of the municipality. The number of human visceral leishmaniasis cases was investigated from Epidemiological Surveillance records. None of the serologically tested dogs showed positive titration. Only two specimens of Lutzomyia neivai, one of Lutzomyia sp. and four of Brumptomyia brumpti specimens were collected. No human visceral leishmaniasis cases were reported. These results suggest that there is no evidence of circulation of L. infantum in Palotina.

Resumo Os cães são os principais reservatórios urbanos da Leishmania infantum, agente causador da leishmaniose visceral (VL), transmitida por vetores conhecidos como flebotomíneos. No Paraná, a primeira detecção de casos positivos caninos ocorreu em 2014, ano em que o Paraná perdeu o status de estado indene. O objetivo deste estudo foi determinar a prevalência da leishmaniose visceral canina no município de Palotina, a ocorrência de vetores que possam transmitir Leishmania infantum e o número de notificação de casos de leishmaniose visceral humana, no período de 2010 a 2020. Para determinar a ocorrência da leishmaniose visceral canina, amostras de sangue de 204 cães foram analisadas, utilizando-se o teste rápido (DPP®) para detectar anticorpos anti-L. infantum. Com o objetivo de investigar a ocorrência de potenciais vetores, coletas foram realizadas mensalmente em 18 pontos na área urbana do município. O número de casos de leishmaniose visceral humana foi investigado a partir de registros da Vigilância Epidemiológica. Nenhum cão testado foi positivo no teste sorológico. Apenas dois espécimes de Lutzomyia neivai, uma de Lutzomyia sp. e quatro de Brumptomyia brumpti foram coletados. Nenhum caso de leishmaniose visceral humana foi notificado. Esses resultados sugerem que não há evidência da circulação de L. infantum em Palotina.

The gene expression of Leishmania infantum chagasi inside Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil

Leishmania infantum chagasi is the causative agent and Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the Americas. We investigated the expression of Leishmania genes within L. longipalpis after artificial infection. mRNAs from genes involved in sugar and amino acid metabolism were upregulated at times of high parasite proliferation inside the insect. mRNAs from genes involved in metacyclogenesis had higher expression in late stages of infection. Other modulated genes of interest were involved in immunomodulation, purine salvage pathway and protein recycling. These data reveal aspects of the adaptation of the parasite to the microenvironment of the vector gut and reflect the preparation for infection in the vertebrate.

Molecular detection of Leishmania spp in Lutzomyia longipalpis in the city of Lavras, Minas Gerais, Brazil

Leishmaniasis is a neglected disease that affects a large part of the world population. Knowing the sand fly fauna of a region is of fundamental importance for guiding health surveillance actions related to the prevention and control of leishmaniasis. A total of 86 specimens of sand flies (60 females and 26 males) were collected. Using the classification proposed by Galati (2003), the following species were identified: Lutzomyia longipalpis (Lutz & Neiva, 1912), Migonemyia migonei (França, 1920), Evandromyia cortelezzi (Brethes, 1923), Ev. sallesi (Galvão & Coutinho, 1939), Nyssomyia whitmani (Atunes & Coutinho, 1939), Psathyromyia lutziana (Costa Lima, 1932), Ev. lenti (Mangabeira, 1938), Brumptomyia sp. (França and Parrot, 1921), and Pressatia sp. (Mangabeira, 1942). Using PCR with internal transcribed spacer target to identify infected sand flies, five Lu. longipalpis females were infected with Leishmania spp. Despite the small number of specimens collected, considerable species diversity was found in the study area.

Ecology of phlebotomine sand flies in an area of leishmaniasis occurrence in the Xakriabá Indigenous reserve, Minas Gerais, Brazil

Abstract INTRODUCTION: Leishmaniasis is a complex vector-borne infectious diseases caused by protozoan parasites in the genus Leishmania and spread by hematophagous phlebotomine sand flies (Diptera: Psychodidae, Phlebotominae). The aim of this study was to investigate the phlebotomine fauna, endophily and exophily of the species found, and possible influence of climatic factors on their populations. METHODS: The study was conducted in the Xakriabá Indigenous Reserve (XIR) in the municipality of São João das Missões in northern Minas Gerais state, Brazil. Insects were collected over three consecutive nights in the last week of each month for 12 months from July 2015 to May 2016 from four houses in four different villages. Two traps were set up in each house: one in the intra-domicile and another in the peri-domicile. RESULTS: A total of 2,012 phlebotomine sand fly specimens representing 23 species and belonging to 10 different genera were captured and identified. Among the studied villages, Riacho do Brejo showed the highest density and diversity of phlebotomine sand flies. The species Lutzomyia longipalpis (80.3%) and Nyssomyia intermedia (7.3%), which are major vectors of visceral and cutaneous leishmaniasis, respectively, had the highest population densities, both in the intra- and peri-domicile. No correlation was observed between climatic factors and the density of phlebotomine sand flies. CONCLUSIONS: The results of the present study may contribute to a better understanding and targeting of the measures for preventing and controlling leishmaniasis by the authorities responsible for indigenous health.

Distribution and ecological aspects of sand flies (Diptera: Psychodidae) species in Northeastern Iran

Objective: To determine both the distribution and the ecological characteristics of sand flies in Golestan Province, northeast of Iran in 2016. Methods: In this study, 34 villages were selected based on their geographical conditions. Sticky paper traps were used for collecting the sand flies. Sampling was carried out in each of villages from May to November. In each village, 60 traps for indoors and 60 for outdoors were monthly installed. The species of all collected sand flies were determined using approved morphological keys. Pearson coefficient correlation was used to find the relationship between the number of collected Phlebotomus papatasi from different villages and incidence rate of zoonotic cutaneous leishmaniasis as well as the number of positive cases of the disease. The altitude of the studied villages was extracted from digital elevation model of the area using GIS and vegetation cover density index of the province was extracted from Modis satellite imagery and distribution map of sand flies drown up. Results: Overall, 5 428 sand flies were collected and identified, belonging to 18 species. Phlebotomus wenyoni was reported for the first time from the area in this study. The frequency of sand flies in the villages located in northeast of the Golestan province (the plateau area, lower altitude, arid and semi-arid climates, and lower vegetation cover density), were more than other villages in this province. There was a significant correlation between the number of collected Phlebotomus papatasi and incidence rate of the zoonotic cutaneous leishmaniasis cases in different villages (r=0.837, P=0.019) as well as the number of positive cases of the disease (r=0.688, P<0.001). Conclusions: In the northeaster areas of Golestan Province which is known as the endemic foci of zoonotic cutaneous leishmaniasis, the abundance of sand flies were more and the conditions for their growth and development were more appropriate.

Distribution and ecological aspects of sand flies (Diptera: Psychodidae) species in Northeastern Iran

Objective:To determine both the distribution and the ecological characteristics of sand flies in Golestan Province, northeast of Iran in 2016.Methods:In this study, 34 villages were selected based on their geographical conditions. Sticky paper traps were used for collecting the sand flies. Sampling was carried out in each of villages from May to November. In each village, 60 traps for indoors and 60 for outdoors were monthly installed. The species of all collected sand flies were determined using approved morphological keys. Pearson coefficient correlation was used to find the relationship between the number of collected Phlebotomus papatasi from different villages and incidence rate of zoonotic cutaneous leishmaniasis as well as the number of positive cases of the disease. The altitude of the studied villages was extracted from digital elevation model of the area using GIS and vegetation cover density index of the province was extracted from Modis satellite imagery and distribution map of sand flies drown up.Results:Overall, 5 428 sand flies were collected and identified, belonging to 18 species. Phlebotomus wenyoni was reported for the first time from the area in this study. The frequency of sand flies in the villages located in northeast of the Golestan province (the plateau area, lower altitude, arid and semi-arid climates, and lower vegetation cover density), were more than other villages in this province. There was a significant correlation between the number of collected Phlebotomus papatasi and incidence rate of the zoonotic cutaneous leishmaniasis cases in different villages (r=0.837, P=0.019) as well as the number of positive cases of the disease (r=0.688, P<0.001).Conclusions:In the northeaster areas of Golestan Province which is known as the endemic foci of zoonotic cutaneous leishmaniasis, the abundance of sand flies were more and the conditions for their growth and development were more appropriate.

Spatial distribution of sand flies (Diptera: Psychodidae; Larroussius group), the vectors of visceral leishmaniasis in Northwest of Iran

Objective: To determine spatial distribution of sand flies (Diptera: Psychodidae; Larroussius group), the vectors of visceral leishmaniasis in Ardabil province, Northwest of Iran. Methods:Sand flies were collected using sticky traps from the 30 selected points in Ardabil province, during May-November 2017. The MaxEnt model in GIS software was used for modeling. Results: A total of 2794 specimens of sand flies were collected, of which 33％ were Larroussius subgenus sand flies. Phlebotomus kandelakii and Phlebotomus wenyoni were the highest and lowest collected species respectively. Based on the modeling, four areas in the province were identified with more than 70％ probability of the presence of Larroussius group vectors which were at risk of visceral leishmaniasis disease transmission. Conclusions: The distribution of Larroussius subgenus sand flies was observed in all parts of Ardabil. But the northern parts of the province (Germi and Bilesavar counties) as well as central part (Ardabil and Meshkinshahr counties) were of great importance in terms of the presence of Larroussius subgenus sand flies and the possibility of transmission of the visceral leishmaniasis.

Spatial distribution of sand flies (Diptera: Psychodidae; Larroussius group), the vectors of visceral leishmaniasis in Northwest of Iran

Objective: To determine spatial distribution of sand flies (Diptera: Psychodidae; Larroussius group), the vectors of visceral leishmaniasis in Ardabil province, Northwest of Iran. Methods: Sand flies were collected using sticky traps from the 30 selected points in Ardabil province, during May-November 2017. The MaxEnt model in GIS software was used for modeling. Results: A total of 2 794 specimens of sand flies were collected, of which 33% were Larroussius subgenus sand flies. Phlebotomus kandelakii and Phlebotomus wenyoni were the highest and lowest collected species respectively. Based on the modeling, four areas in the province were identified with more than 70% probability of the presence of Larroussius group vectors which were at risk of visceral leishmaniasis disease transmission. Conclusions: The distribution of Larroussius subgenus sand flies was observed in all parts of Ardabil. But the northern parts of the province (Germi and Bilesavar counties) as well as central part (Ardabil and Meshkinshahr counties) were of great importance in terms of the presence of Larroussius subgenus sand flies and the possibility of transmission of the visceral leishmaniasis.

Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania and sand flies co-exist.

Description of a new species of Psathyromyia Barreto (Diptera, Psychodidae, Phlebotominae) from Amazonas state, Brazil

ABSTRACTA new species of phlebotomine sand fly is described and illustrated based on the male and female morphological characters of specimens collected from Tefé and Coari municipalities, Amazonas state. The phlebotomine sand flies were collected with CDC light traps used as aspirators at the base of tree trunks. Both male and female specimens collected in Tefé municipality were first identified as Psathyromyia souzacastroi. After the analysis of the holotype of Pa. souzacastroi deposited in Smithsonian Institute/Walter Reed Biosystematics Unit, it was observed that the morphotypes collected in Tefé municipality belong to a distinct species, which characterization is here presented.

Perfil de transcrição gênica de células humanas estimuladas com a saliva de Lu. intermedia ou de Lu / Gene transcription profile of human cells stimulated with Lu saliva. Intermedia or Lu

A saliva dos flebotomíneos transmissores do parasita Leishmania possui uma variedade de agentes farmacológicos, como anticoagulantes, vasodilatadores além de moléculas imunomoduladoras e anti-inflamatórias. A saliva de Lu. intermedia e de Lu. longipalpis provocam o aumentam da infecção por diferentes espécies de Leishmania, em modelos experimentais.Entretanto a pré-exposição à saliva de Lu. longipalpis confere proteção a infecção, enquanto a pré-exposição à saliva de Lu. intermedia causa exacerbação da doença. Neste trabalho estimulamos as células do sangue de voluntários sadios com a saliva de Lu. intermedia ou de Lu. longipalpis e, posteriormente, o RNA foi extraído e utilizado no sequenciamento em larga escala (RNAseq). O estudo demonstrou que a saliva de Lu. intermedia e de Lu.longipalpismodula a expressão de uma série de genes e os processos biológicos mais requentes são semelhantes após a estimulação com as duas diferentes salivas. Identificamos seis processos biológicos comuns às salivas dos dois lebotomineos:Taxis,Chemotaxis,Locomotory behavior, Positive regulation of immune system process, Regulation of cytokine production e Regulation of cell activation. Dentre os genes que caracterizam esses seis processos,detectamosgenes que codificam quimiocinas, citocinas além de moléculas de superfície tais como CCL19, CCL2, CCL3, CCL4, CD80, CD83, IL10, IL1B, IL4, IL6.Definimos um conjunto de genes encontrados exclusivamente na amostra estimulada com a saliva de Lu. intermedia (ADA, CCL23, CCL3, CCL3L1, CXCL11, PDGFB, PDPN, PLAU e TNFSF15). Da mesma forma, encontramos um outro conjunto de genes expresso exclusivamenteem células estimuladas com a saliva de Lu. longipalpis(ENPP2, EREG, IDO1,IL1A e VEGFA). Essas diferenças podem fornecer pistas para o desenvolvimento da leishmaniose em indivíduos continuamente expostos à saliva de Lu. intermedia.

The saliva of sand flies, vectors of leishmania parasite, has a variety of pharmacological agents, such as anticoagulants, vasodilators as well as immunomodulatory and antiinflammatory molecules. Lu. intermedia and Lu. longipalpis increase the infection by different species of leishmania in experimental models. However, the pre-exposure to Lu. longipalpis saliva provides protection to infection, while the pre-exposure Lu. intermedia saliva causes exacerbation of the disease. In this work, we stimulated peripheral blood cells from healthy volunteers with salivary glands from Lu. intermedia or Lu. longipalpis and later RNA was extracted and used in large-scale sequencing (RNAseq). This study demonstrated that Lu. intermedia and Lu. longipalpis saliva modulate the expression of a number of genes and the most frequent biological processes are similar in cells stimulated with both saliva. We identified six biological processes commonly upregulated following stimulation with saliva of the two sand flies: taxis, chemotaxis, locomotory behavior, positive regulation of immune system process, regulation of cytokine production and regulation of cell activation. Among the genes that characterize these six biological processes, we detected genes encoding chemokines, cytokines plus surface molecules such as:CCL19, CCL2, CCL3, CCL4, CD80, CD83, IL10, IL1B, IL4 and IL6. We found a set of genes upregulated exclusively in cells stimulated with Lu. intermedia saliva (ADA, CCL23, CCL3, CCL3L1, CXCL11, PDGFB, PDPN, PLAU and TNFSF15). Similarly, another set of genes was expressed only in cells stimulated Lu. longipalpis saliva (ENPP2, EREG, IDO1,IL1A and VEGFA). These differences may provide clues for the development of leishmaniasis in individuals continuously exposed to Lu. intermedia saliva.

DISTINCT CELLULAR MIGRATION INDUCED BY Leishmania infantum chagasi AND SALIVA FROM Lutzomyia longipalpis IN A HEMORRHAGIC POOL MODEL / Migração celular distinta induzida por Leishmania infantum chagasi e saliva de Lutzomyia longipalpis em um modelo de pool hemorrágico

Recruitment of a specific cell population after Leishmania infection can influence the outcome of the disease. Cellular migration in response to Leishmania or vector saliva has been reported in air pouch model, however, cellular migration induced by Leishmania associated with host's blood and vector saliva in this model has not been described. Herein we investigated cellular migration into air pouch of hamster after stimulation with combination of L. chagasi and host's blood and Lutzomyia longipalpis saliva. Migration induced by saliva was 3-fold more than those induced by L. chagasi alone. Additionally, L. chagasi associated with blood and saliva induced significantly even more leukocytes into air pouch than Leishmania alone. L. chagasi recruited a diverse cell population; however, most of these cells seem to have not migrated to the inflammatory exudate, remaining in the pouch lining tissue. These results indicate that L. chagasi can reduce leukocyte accumulation to the initial site of infection, and when associated with vector saliva in the presence of blood components, increase the influx of more neutrophils than macrophages, suggesting that the parasite has developed a strategy to minimize the initial inflammatory response, allowing an unlimited progression within the host. This work reinforces the importance of studies on the salivary components of sand fly vectors of leishmaniasis in the transmission process and the establishment of the infection.

O recrutamento de uma população de células específicas após infecção por Leishmania pode influenciar o resultado da doença. A migração celular em resposta a Leishmania ou saliva do vetor tem sido reportada utilizando o modelo da bolsa de ar subcutânea, entretanto, a migração celular induzida por Leishmania associada com o sangue do hospedeiro e saliva do vetor neste modelo ainda não foi descrita. Neste trabalho foi investigada a migração celular no modelo da bolsa de ar subcutânea em hamster após a estimulação com a combinação de L. chagasi, sangue do hospedeiro e saliva de Lutzomyia longipalpis. A migração induzida por saliva foi três vezes maior do que a induzida por L. chagasi sozinha. Adicionalmente, L. chagasi associada com sangue e saliva induziu significativamente ainda mais leucócitos no exsudato inflamatório do que o estímulo com Leishmania sozinha. L. chagasi recrutou uma população de células distintas, no entanto, a maioria dessas células parece não ter migrado para o exsudato inflamatório, permanecendo no tecido da bolsa de ar. Estes resultados indicam que L. chagasi pode reduzir o acúmulo de leucócitos para o local inicial da infecção e que quando associada à saliva do vetor e na presença de componentes do sangue aumenta o influxo de mais neutrófilos do que macrófagos, sugerindo que o parasito desenvolveu uma estratégia para minimizar a resposta inflamatória inicial, permitindo uma progressão ilimitada dentro do hospedeiro. Este trabalho reforça a importância de mais estudos sobre os componentes da saliva dos vetores das leishmanioses no processo de transmissão e no estabelecimento da infecção.

Sand Fly Fauna (Diptera, Pcychodidae, Phlebotominae) in DifferentLeishmaniasis- Endemic Areas of Ecuador, Surveyed by Using a Newly Named Mini-Shannon Trap

To study the sand fly fauna, surveys were performed atfour different leishmaniasis-endemic areas of Ecuador, during February 2013 andApril 2014. The conventional Shannon trap was modified and simplified to anewly named mini-Shannon trap for its multiple uses at different study sites,such as limited, forested and narrow spaces. The mini-Shannon, CDC light trapsand the protected human landing method were employed for sand fly collection.The species identification of sand flies was performed mainly based on themorphology of spermathecae and cibarium, after dissection of freshsamples.  In this study, therefore, onlyfemale samples were used for analysis. A total of1,480 female sand flies belonging to 25<i> Lutzomyia</i> species were collected. Numbers of the female sand fliescollected by each trap were 417 (28.2%) by the mini-Shannon trap, 259 (17.5%)by CDC light trap and 804 (54.3%) by human landing. The total number of sand flies per trap collected bydifferent methods was markedly affected by study sites, probably because ofdifferent species compositions at each locality. Further, as an additionalstudy, the attractiveness of sand flies against the mini-Shannon traps poweredwith LED White-light and LED Black-light, waspreliminary tested, together with CDC light trap and human landing. In the test,a total of 426 sand flies of nine <i>Lutzomyia</i> species, seven man-biting and two non-man-biting species, were collected by threecapture trials during May and June 2014 in an area endemic for leishmaniasis(La Ventura). The Black-light equipped trap was relatively superior in capturenumbers to the White-light equipped one, but no significant difference wasobserved statistically between the two traps.

Sand Fly Fauna (Diptera, Pcychodidae, Phlebotominae) in Different Leishmaniasis-Endemic Areas of Ecuador, Surveyed Using a Newly Named Mini-Shannon Trap

To study the sand fly fauna, surveys were performed at four different leishmaniasis-endemic sites in Ecuador from February 2013 to April 2014. A modified and simplified version of the conventional Shannon trap was named “mini-Shannon trap” and put to multiple uses at the different study sites in limited, forested and narrow spaces. The mini-Shannon, CDC light trap and protected human landing method were employed for sand fly collection. The species identification of sand flies was performed mainly based on the morphology of spermathecae and cibarium, after dissection of fresh samples. In this study, therefore, only female samples were used for analysis. A total of 1,480 female sand flies belonging to 25 <i>Lutzomyia</i> species were collected. The number of female sand flies collected was 417 (28.2%) using the mini-Shannon trap, 259 (17.5%) using the CDC light trap and 804 (54.3%) by human landing. The total number of sand flies per trap collected by the different methods was markedly affected by the study site, probably because of the various composition of species at each locality. Furthermore, as an additional study, the attraction of sand flies to mini-Shannon traps powered with LED white-light and LED black-light was investigated preliminarily, together with the CDC light trap and human landing. As a result, a total of 426 sand flies of nine <i>Lutzomyia</i> species, including seven man-biting and two non-biting species, were collected during three capture trials in May and June 2014 in an area endemic for leishmaniasis (La Ventura). The black-light proved relatively superior to the white-light with regard to capture numbers, but no significant statistical difference was observed between the two traps.

Estimación del tiempo de desarrollo de Lutzomyia evansi bajo condiciones experimentales / Estimation of development time for Lutzomyia evansi under experimental conditions

Objetivo: Estimar el tiempo promedio de desarrollo de Lutzomyia evansi. Materiales y métodos: Se inició una colonia de Lutzomyia evansi con individuos recolectados en la zona urbana de la ciudad de Sincelejo (Colombia). La colonia fue mantenida en el laboratorio durante tres generaciones filiales bajo condiciones experimentales promedio de 26°C de temperatura y 94% de humedad relativa. Resultados: La duración del desarrollo de Lutzomyia evansi fue de 36 a 45 días. El tiempo requerido para el desarrollo de los huevos fue en promedio de 6,75 días (rango de 6 a 8 días). La duración en promedio de los diferentes estadios larvales fue 5,75 días en larvas de primer estadio (rango de 5 a 8 días), 5,75 días en larvas de segundo estadio (rango de 4 a 7 días), 5 días en larvas de tercer estadio (rango de 4 a 7 días) y 7 días en larvas de cuarto estadio (rango e 6 a 8 días). En la fase de pupa, la duración en promedio fue de 9,75 días (rango de 7 a 17 días). Conclusiones: El tiempo promedio requerido para el desarrollo de Lutzomyia evansi, comprendido desde la alimentación sanguínea de la hembra madre hasta la emergencia del adulto, es de 40 días.

Objective: To estimate the mean development time for Lutzomyia evansi. Materials and methods: A laboratory colony of Lutzomyia evansi was started from sand flies collected in the urban area of the City of Sincelejo, Colombia. The colony was main-tained during three filial generations under experimental conditions of 26°C of mean tem-perature, and 94% of average relative humidity. Results: The duration of the development of Lutzomyia evansi was from 36 to 45 days. The development time for eggs was, on average, 6, 75 days (interval from 6 to 8 days). The mean duration of the different larval instars was 5,75 days in first instar (interval from 5 to 8 days), 5,75 days in second instar (interval from 4 to 7 days), 5 days in third instar (in-terval from 4 to 7 days) and 7 days in fourth instar (interval from 6 to 8 days). In the stage of pupa the development time was, on average, 9, 75 days (interval from 7 to 17 days). Conclusions: The mean development time for Lutzomyia evansi, from the female's blood meal to adult emergence, is 40 days.

A study on delay in treatment of kala-azar patients in Bangladesh.

Visceral leishmaniasis (kala-azar) continues to be a major rural public health problem in Bangladesh. A cross-sectional study was carried out in two subdistricts of Mymensingh district from January 2006 to June 2007 to evaluate the delay kala-azar treatment. Suspected patients who attended to out patient department (OPD) were subjected to a dipstick test (RK39) for kala-azar. Sixty five from Bhaluka and 60 positive patients from Gafargaon subdistrict were enrolled. Most of the patients (80%) first visited nonqualified private practitioners, while only 15.2% consulted registered doctors. Fifty per cent were referred to the Upazilla health complex (UZHC) by the family members or relatives. About 49% and 43% patients required third and second health-care providers for kala-azar treatment, respectively. Patient delay ranged from 2 to 30 days; median 4 (IQR 3 to 7 days), the system delay ranged from 0 days to 225 days; median 54 (IQR 40–66 days). Residential status (p value <0.05) had impact on patient delay. Educational status and number of treatment providers had impact on system delay (p<0.05). System delay rather than patient delay is the important weakness of the kala-azar control programme in Bangladesh. Residence in rural areas, low educational background and treatment providers are associated with these delays. A proper educational programme may reduce the delay.

Genetic differentiation in natural populations of Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae) with different phenotypic spot patterns on tergites in males

Entomological surveys in the state of Maranhão have recorded morphologically distinct populations of Lutzomyia longipalpis (Lutz & Neiva). Some populations have one pair of spots (1S) on the fourth tergite, while others have two pairs (2S) on the third and fourth tergites of males. In the present study we investigated the degree of genetic polymorphism among four populations in the municipalities of Caxias, Codó and Raposa, in the state of Maranhão, Brazil, by using RAPD (Random Amplified Polymorphic DNA) markers. A total of 35 loci were identified, of which 30 were polymorphic. The highest polymorphism was observed with primer OPA 4, which produced 11 different profiles. Genetic diversity was assessed using grouping methods that produced a dendrogram in which the genotypes could be clearly separated into two main clades according to the number of spots on the male abdominal tergites. One cluster contained the populations from Caxias and Codó, and the other was formed by the populations from Raposa and Codó. The results of our RAPD analysis showed a clear separation between the populations with one and two pairs of spots. The epidemiologic significance of this genetic differentiation should be investigated in future studies.

Bionomics of phlebotomine sand flies (Diptera: Psychodidae) in the province of Al-Baha, Saudi Arabia

The bionomics of phlebotomine sand flies (Diptera: Psychodidae) were studied for two successive years (January 1996-December 1997) at 12 collecting stations representing six sectors of the province of Al-Baha, Saudi Arabia. The predominant species was Phlebotomus bergeroti (41.7 percent), followed by lesser numbers of Phlebotomus sergenti (11 percent), Phlebotomus arabicus (10.6 percent), Sergentomyia tiberiadis (10.5 percent), Phlebotomus papatasi (10.2 percent), Sergentomyia antennata (9.6 percent), Phlebotomus alexandri (3 percent), Phlebotomus orientalis (2.3 percent) and Sergentomyia clydei (1.1 percent). The distribution of the collected species including species that are elsewhere known to act as vectors of human cutaneous leishmaniasis were distributed across different altitudes in Al-Baha. P. bergeroti, P. papatasi and P. arabicus were more abundant indoors; however, P. sergenti was more abundant outdoors. Sand fly populations exhibited three patterns of seasonal abundance in terms of their monthly activity. P. bergeroti, P. sergenti and P. arabicus were found to be naturally infected with Leishmania-like flagellates at an infection rate of 0.2 percent.

Primeiro registro de Lutzomyia araracuarensis (Morales & Minter) (Diptera: Psychodidae) no Brasil / First Report of Lutzomyia araracuarensis (Morales & Minter) (Diptera: Psychodidae) in Brazil

A male of Lutzomyia araracuarensis (Morales & Minter) and possibly six females of this same species were found in the northeastern area of Manacapuru county, Amazonas State. Samples were collected using light traps CDC, from April 2003 to June 2004.