Vector Of Leishmaniasis Research Articles

Richness and Diversity of Phlebotomine Sand Flies (Diptera: Psychodidae) in North Khorasan Province, Northeast of Iran

We aimed to determine the species composition, richness and diversity indices of the Phlebotomine sand flies at different topographic condition in visceral (VL) and cutaneous leishmaniasis (CL) foci in the northeast of Iran. This cross-sectional study was conducted during 2016 in North Khorasan Province. The sampling was focused on rural regions, where human cases of VL and/or CL were diagnosed and reported during last 5 years. Sand flies were collected three times each twenty days during peak periods of seasonal activity. Seven collection methods were used. Some Alpha and Beta diversity indices were calculated. Overall, 7253 sand flies were collected and identified. They were from 19 species of Phlebotominae sand-flies. Phlebotomus sergenti and Ph. papatasi were the most prevalent (84.9%) species in the study area. Species richness (S) was very different in three areas and were18, 8, and 4 respectively but Evenness (E) were 0.357, 0.345, and 0.380, so evenness was almost equal in the study areas. Shannon Index (H) and Margalef Richness Index were calculated 1.033, 0.718, 0.527 and 2.117, 0.8998, 0.4006 respectively. The sand fly fauna in North Khorasan Province was very rich and often included some of the most important proven or suspected vectors of leishmaniasis. Species diversity indices (Shannon index, and Simpson's index) were not high due to decreasing in evenness. The Margalef richness index could accurately reflect the biodiversity of sand flies between three subtidal locations.

Revisiting an old idea: engineering against vector-borne diseases in the domestic environment.

On 21 April 1983 the Royal Society of Tropical Medicine and Hygiene held a joint meeting with the Institute of Civil Engineers at Manson House on ‘Engineering against Insect-borne Diseases in the Domestic Environment’. The summary of a talk by Chris Schofield and Graham White on ‘House design and domestic vectors of disease’ was published in a special issue of the Transactions of the Royal Society of Tropical Medicine and Hygiene.1 The manuscript highlights the home and peri-domestic environment as an important site of transmission for many vector-borne diseases, due to the presence of people, and in some cases animals, on which to feed, and provision of shelter from predators and extreme climate. For example, malaria mosquito vectors such as Anopheles gambiae readily enter houses at night to feed on humans. Aedes aegypti, the mosquito vector of diseases including dengue, yellow fever, Zika and chikungunya, is common in urban areas where water that collects in discarded plastic containers, car tires and water storage containers provides ideal aquatic habitats for this mosquito to lay its eggs. Cracked and uneven floors and walls can provide habitats for flea larvae, house dust mites, sandflies and triatomine bugs; the latter are vectors of leishmaniasis and Chagas disease, respectively. Flooded pit latrines, cracked septic tanks and stormwater drains provide habitats for Culex mosquitoes, which can transmit filariasis and contribute to nuisance biting.

Machine learning approaches in GIS-based ecological modeling of the sand fly Phlebotomus papatasi, a vector of zoonotic cutaneous leishmaniasis in Golestan province, Iran

The distribution and abundance of Phlebotomus papatasi, the primary vector of zoonotic cutaneous leishmaniasis in most semi-/arid countries, is a major public health challenge. This study compares several approaches to model the spatial distribution of the species in an endemic region of the disease in Golestan province, northeast of Iran. The intent is to assist decision makers for targeted interventions. We developed a geo-database of the collected Phlebotominae sand flies from different parts of the study region. Sticky paper traps coated with castor oil were used to collect sand flies. In 44 out of 142 sampling sites, Ph. papatasi was present. We also gathered and prepared data on related environmental factors including topography, weather variables, distance to main rivers and remotely sensed data such as normalized difference vegetation cover and land surface temperature (LST) in a GIS framework. Applicability of three classifiers: (vanilla) logistic regression, random forest and support vector machine (SVM) were compared for predicting presence/absence of the vector. Predictive performances were compared using an independent dataset to generate area under the ROC curve (AUC) and Kappa statistics. All three models successfully predicted the presence/absence of the vector, however, the SVM classifier (Accuracy = 0.906, AUC = 0.974, Kappa = 0.876) outperformed the other classifiers on predicting accuracy. Moreover, this classifier was the most sensitive (85%), and the most specific (93%) model. Sensitivity analysis of the most accurate model (i.e. SVM) revealed that slope, nighttime LST in October and mean temperature of the wettest quarter were among the most important predictors. The findings suggest that machine learning techniques, especially the SVM classifier, when coupled with GIS and remote sensing data can be a useful and cost-effective way for identifying habitat suitability of the species.

Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9.

BackgroundClustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand flies, the vectors of Leishmaniasis.Methodology/Principal findingsHere, we described a detailed protocol for sand fly embryo microinjection taking into consideration the sand fly life cycle, and manipulation and oviposition requirements of this non-model organism. Following our microinjection protocol, a hatching rate of injected embryos of 11.90%-14.22% was achieved, a rate consistent with other non-model organism dipterans such as mosquitoes. Essential factors for the adaptation of CRISPR/Cas9 technology to the sand fly field were addressed including the selection of a target gene and the design and production of sgRNA. An in vitro cleavage assay was optimized to test the activity of each sgRNA and a protocol for Streptococcus pyogenes Cas9 (spCas9) protein expression and purification was described. Relevant considerations for a successful gene editing in the sand fly such as specifics of embryology and double-stranded break DNA repair mechanisms were discussed.Conclusion and significanceThe step-by-step methodology reported in this article will be of significant use for setting up a sand fly embryo microinjection station for the incorporation of CRISPR/Cas9 technology in the sand fly field. Gene editing strategies used in mosquitoes and other model insects have been adapted to work with sand flies, providing the tools and relevant information for adapting gene editing techniques to the vectors of Leishmaniasis. Gene editing in sand flies will provide essential information on the biology of these vectors of medical and veterinary relevance and will rise a better understanding of vector-parasite-host interactions.

Establishment of a method for Lutzomyia longipalpis sand fly egg microinjection: The first step towards potential novel control strategies for leishmaniasis

Leishmaniasis is a vector-borne parasitic disease transmitted by sand flies that affects 1.3 million people across 98 countries, with limited control strategies due to the lack of an available vaccine and the emergence of insecticide resistance. Novel control strategies that are being explored for mosquito-borne diseases, such as Wolbachia bacterial inhibition of pathogens and genetically modified insects (e.g. using CRISPR-Cas9 editing), rely on the ability to consistently inject embryos of the target species. Here we present a novel method to obtain and inject preblastoderm sand fly embryos of the genus Lutzomyia (Lu.)longipalpis, the principle vector of zoonotic visceral leishmaniasis in South America. The procedures required to obtain sufficiently young Lu. longipalpis colony embryos are described alongside a microinjection technique that permits rapid injection and minimal handling of small sand fly embryos post-injection. Using a strain of Wolbachia as a 'marker' for successful injection, our protocol produced early generation Wolbachia transinfected Lu. longipalpis lines, demonstrating its potential as the first step for use in novel applied strategies for sand fly control.

Ecological niche modeling predicting the potential distribution of Leishmania vectors in the Mediterranean basin: impact of climate change

BackgroundDue to climate change, the geographical distribution of sand flies during the last decades has shifted northward from latitudes below 45°N in southern Europe to latitudes just above 50○N. Recent studies show that some phlebotomine sand flies were recorded in several parts of Germany and Belgium. In central Europe, some autochthone leishmaniasis cases are being recorded in regions traditionally regarded as leishmaniasis-free. An important challenge is to predict the geographical distribution of leishmaniasis vectors under new climatic conditions. In this study, we attempted to predict the current distribution of six leishmaniasis vectors in the Mediterranean basin and forecast species’ geographical shift under future climate scenarios using an ensemble ecological niche modeling approach. Species records were obtained from scientific surveys published in the research literature between 2006 and 2016. A series of climate metrics describing temperature and precipitation in the study area under two climatic scenarios were obtained from WorldClim database. A consensus model was derived from six varieties of modeling approaches (regression, machine learning and classification techniques) in order to ensure valid prediction of distribution of vectors under different climate scenarios.ResultsModel performance was generally high for the included species with a specificity (true negative rate) ranging from 81.03 to 96.52% (mean = 86.94%) and a sensitivity (true positive rate) ranging from 87.93 to 100% (mean = 96.98%). Our work evidenced the hypothesis of the widespread of Leishmania vectors under climate change scenarios. All of the studied species are prospected to gain new areas that are actually not suitable for vectors’ survival. Phlebotomine sand flies are prospected to invade extra-Mediterranean regions, especially western and central Europe.ConclusionsOur study confirmed the importance of environmental and climate factors on the distribution of leishmaniasis vectors and demonstrated the performance of ecological niche modeling in the prediction of the geographical spread of vector-borne diseases. Ecological niche modeling should be considered in the future as a valuable tool in addition to experimental laboratory studies for a better understanding of the biology of vector species.

Susceptibility of field-collected Phlebotomus argentipes (Diptera: Psychodidae) sand flies from Bangladesh and Nepal to different insecticides

BackgroundThe sand fly Phlebotomus argentipes is the vector for visceral leishmaniasis (VL) in the Indian sub-continent. In Bangladesh since 2012, indoor residual spraying (IRS) was applied in VL endemic areas using deltamethrin. In Nepal, IRS was initiated in 1992 for VL vector control using lambda-cyhalothrin. Irrational use of insecticides may lead to vector resistance but very little information on this subject is available in both countries. The objective of this study was to generate information on the susceptibility of the vector sand fly, P. argentipes to insecticide, in support of the VL elimination initiative on the Indian sub-continent.MethodsSusceptibility tests were performed using WHO test kits following the standard procedures regarding alpha cypermethrin (0.05%), deltamethrin (0.05%), lambda-cyhalothrin (0.05%), permethrin (0.75%), malathion (5%) and bendiocarb (0.1%) in six upazilas (sub-districts) in Bangladesh. In Nepal, the tests were performed for two insecticides: alpha cypermethrin (0.05%) and deltamethrin (0.05%). Adult P. argentipes sand flies were collected in Bangladesh from six VL endemic upazilas (sub-districts) and in Nepal from three endemic districts using manual aspirators.ResultsThe results show that VL vectors were highly susceptible to all insecticides at 60 minutes of exposure in both countries. In Bangladesh, corrected mortality was 100% at 15 minutes as well as 30 minutes of exposure. The study sites in Nepal, however, showed some diverse results, with a mortality rate less than 90% for 15 minutes of exposure with alpha cypermethrin and deltamethrin in two districts but was above 95% after 30 minutes of exposure.ConclusionsThese results suggest that the insecticides tested can still be used in the national programmes of Bangladesh and Nepal. However, insecticide rotation should be performed to mitigate the possible development of insecticide resistance. Periodic susceptibility tests should be performed by the countries to get timely alerts regarding insecticide resistance.

Impact of meteorological and geographical factors on the distribution of Phlebotomus chinensis in northwestern mainland China.

Phlebotomine sandflies (Diptera: Phlebotomidae) are vectors of the zoonotic disease leishmaniasis. To better understand the distribution of phlebotomine sandflies in order to facilitate control of leishmaniasis transmission, the present study explored the impacts of climate and landscape on local abundances of Phlebotomus chinensis in northwestern mainland China. Identification records were used to create a geodatabase for the locations at which P.chinensis had been collected in the region, and a regional-scale map was developed to show the distribution of P.chinensis. Location data and data on environmental factors during the years in which the samples were collected were incorporated, and a presence-only modelling method was used to evaluate the species' habitat preferences and to predict its potential distribution in northwestern mainland China. Jackknife analysis revealed that several meteorological variables, including maximum temperature in the warmest quarter, precipitation in the driest month, daily average temperature and daily precipitation, significantly affected the presence of this species. Moreover, the presence of P.chinensis was significantly associated with grassland and shrubland. Probability distributions using maximum entropy were used to map the distribution ranges of P.chinensis based on suitable habitats in northwestern mainland China. The models generated can be used to develop detailed strategies for the prevention and control of leishmaniasis.

Entomological surveys of Lutzomyia flaviscutellata and other vectors of cutaneous leishmaniasis in municipalities with records of Leishmania amazonensis within the Bragança region of Pará State, Brazil.

In southeast Amazon, Lutzomyia (Nyssomyia) flaviscutellata is the incriminated vector of Leishmania (Leishmania) amazonensis, a causative agent of zoonotic cutaneous leishmaniasis (CL). The optimal methods for surveying Lu. flaviscutellata were investigated in the Bragança region, northeast Pará State, Brazil, selected for the presence of Le. amazonensis. The performances of modified Disney traps and CDC light traps were compared in four ecotopes within and around four village transects during the wet and dry seasons. The physiological age of female sand flies was estimated and natural infection by flagellates was evaluated by dissection. Disney traps were better for detecting the presence of Lu. flaviscutellata, while CDC traps performed well for detecting Lutzomyia (Nyssomyia) antunesi, suspected vector of Leishmania lindenbergi. The former was more abundant during the wet season, when female flies were naturally infected with Le. amazonensis. These findings identified the environments of local transmission. In order to improve surveys of Lu. flaviscutellata as part of integrated epidemiological surveillance of CL, our recommendations include focusing vector surveys with Disney traps on forest fragments where people work, during the seasonal peak of the vector. Further field studies are required to make model-based predictions of seasonal variations in the vectorial capacity of vector populations.

Establishment of a method for Lutzomyia longipalpis sand fly embryo microinjection: The first step towards potential novel control strategies for leishmaniasis

Leishmaniasis is a vector-borne parasitic disease transmitted by sand flies that affects 1.3 million people across 98 countries, with limited control strategies due to the lack of an available vaccine and the emergence of insecticide resistance. Novel control strategies that are being explored for mosquito-borne diseases, such as Wolbachia bacterial inhibition of pathogens and genetically modified insects (e.g. using CRISPR-Cas9 editing), rely on the ability to consistently inject embryos of the target species. Here we present a novel method to obtain and inject preblastoderm sand fly embryos of the genus Lutzomyia (Lu.) longipalpis, the principle vector of zoonotic visceral leishmaniasis in South America. The procedures required to obtain sufficiently young Lu. longipalpis colony embryos are described alongside a microinjection technique that permits rapid injection and minimal handling of small sand fly embryos post-injection. Using a strain of Wolbachia as a ‘marker’ for successful injection, our protocol produced early generation Wolbachia transinfected Lu. longipalpis lines, demonstrating its potential as the first step for use in novel applied strategies for sand fly control.

Detection of Pyrethroid Resistance Mutations in the Major Leishmaniasis Vector Phlebotomus papatasi.

Phlebotomine sand flies (Diptera: Psychodidae) are primary vectors of leishmaniasis. Greece and Turkey are both endemic for visceral and cutaneous leishmaniasis and are widely affected by the disease. Measures commonly applied for controlling sand flies rely on the use of insecticides, predominantly pyrethroids. A worldwide problem associated with the intensive use of insecticides is the development of resistance. Scarce information is available regarding the resistance status in sand fly populations. Sand flies were collected from Greece (Thessaloniki, Peloponnese, Chios island) and Turkey (Sanliurfa) and analyzed for the presence and frequency of target-site knockdown resistance mutations on the voltage-gated sodium channel (Vgsc) gene. Five sand fly species were included in the analysis: Phlebotomus perfiliewi Parrot, Phlebotomus neglectus Tonnoir, Phlebotomus simici Nitzulescu, Phlebotomus tobbi Adler and Theodor, and Phlebotomus papatasi Scopoli. Their Vgsc gene-domain II was analyzed for the presence of known pyrethroid resistance mutations. The mutation 1014F, associated with pyrethroid-resistant phenotypes, was detected in P. papatasi sand flies from Sanliurfa at an allele frequency of 48%. Homozygotes for the wild type allele 1014L (Leu/Leu) represented 36% of the population, while homozygotes for the resistant allele 1014F (Phe/Phe) and heterozygotes encompassing both alleles (Leu/Phe) each had a frequency of 32%. In all other sand fly species, only the wild type allele 1014L was detected. This is the first report for the detection of resistance mutations in the major leishmaniasis vector P. papatasi and is of major concern regarding leishmaniasis control.

Wsp-based analysis of Wolbachia strains associated with Phlebotomus papatasi and P. sergenti (Diptera: Psychodidae) main cutaneous leishmaniasis vectors, introduction of a new subgroup wSerg

Sand flies of Phlebotomus papatasi and P. sergenti are the main vectors of cutaneous leishmanisis (CL) in the old world. We aimed to screen Iranian P. papatasi and P. sergenti for their natural infections with Wolbachia and to determine their phylogenetic association with other species. Wolbachia surface protein (wsp) gene was PCR amplified from DNA extracted from Phlebotomus species, sequenced, and were analysed in combination with wsp sequences related to Phelebtominae and other insects. All Wolbachia-infecting Iranian sand flies of P. papatasi and P. sergenti were classified in the Supergroup A., Wolbachia isolated from P. sergenti were clustered in a new subgroup within Supergroup A so-called wSreg. The Wolbachia strains identified from the P. papatasi clustered mainly in the subgroup wPap and partly in wSerg. Multiple Wholbachia infection within a single population of P.papatasi warrants investigation on existence and intensity of cytoplasmic incompatibility between the wPap and wSerg subgroups.

Composition of sand fly fauna (Diptera: Psychodidae) and detection of Leishmania DNA (Kinetoplastida: Trypanosomatidae) in different ecotopes from a rural settlement in the central Amazon, Brazil

BackgroundPhlebotomine sand flies (Diptera: Psychodidae) are vectors of Leishmania species, the etiological agents of leishmaniasis, which is one of the most important emerging infectious diseases in the Americas. In the state of Amazonas in Brazil, anthropogenic activities encourage the presence of these insects around rural homes. The present study aimed to describe the composition and distribution of sand fly species diversity among the ecotopes (intradomicile, peridomicile and forest) in an area of American cutaneous leishmaniasis transmission and detect natural infection with Leishmania DNA to evaluate which vectors are inside houses and whether the presence of possible vectors represents a hazard of transmission.ResultsPhlebotomine sand flies were collected using light traps. A total of 2469 specimens representing 54 species, predominantly females (71.2%), were collected from four sites. Polymerase chain reaction analysis was performed on 670 samples to detect Leishmania DNA. Most of the samples (79.5%) were collected in the forest, with areas closer to rural dwellings yielding a greater abundance of suspected or proven vectors and a larger number of species containing Leishmania DNA. Nyssomyia umbratilis and Bichromomyia flaviscutellata were found near rural homes, and Ny. umbratilis was also found inside homes. Leishmania DNA was detected in different species of sand flies in all ecotopes, including species with no previous record of natural infection.ConclusionsThere is no evidence that vectors of American cutaneous leishmaniasis are becoming established inside homes, but there are sand flies, including Ny. umbratilis and other possible vectors, in environments characterized by a human presence. These species continue to be predominant in the forest but are prevalent in areas closer to ecotopes with a greater human presence. The existence of proven or suspected vectors in this ecotope is due to the structural organization of rural settlements and may represent a hazard of transmission. Although the detection of Leishmania DNA in species that were not previously considered vectors does not mean that they are transmitting the parasite, it does show that the parasite is circulating in ecotopes where these species are found.

Kala-azar in Darfur: Evidence for indigenous transmission in Al-Malha Locality, North Darfur, western Sudan

BackgroundRecent reports showed high numbers of visceral leishmaniasis cases in North Darfur, western Sudan. Due to a lack of previous studies, no information is available on local transmission of the disease in these areas. Therefore, a pilot entomological and epidemiological study was conducted in Al-Malha Locality during the year 2013, to investigate possibility of local transmission and places and times of the year where and when people contract the infection.MethodsKala-azar incidence data were obtained from records of Ministry of Health, North Darfur; Al-Malha rural hospital; and the Federal Ministry of Health, Division of Communicable and Non-communicable Diseases. Sand flies were collected using sticky paper and rodent burrow traps from five different microhabitats during three different phases of the year. Species identification was undertaken using appropriate taxonomic keys. Data were statistically analyzed to determine the distribution of kala-azar among different age groups and between sexes, and to compare the species richness and distribution of different sandfly species between the different microhabitats.ResultsThe most affected age groups with kala-azar during the period 2013–2016 were children between one and five years old and those under one year. Females were found to be more affected than males. A total of 918 sand fly specimens were collected using sticky paper and rodent burrow traps from five microhabitats. Identified specimens belong to 13 species; 5 Phlebotomus and 8 Sergentomyia. Phlebotomus orientalis, the principal vector of visceral leishmaniasis (VL) in Sudan and other East African countries, was found for the first time in the area. No other known vector of VL was found in the collection. The highest number of sand flies was recorded during the summer season (63%), with S. antennata (48%) and S. schwetzi (24.1%) being the most abundant species. Among Phlebotomus species, P. orientalis showed relatively high density (8.6%). A dry seasonal water course (called “Khor”) seems to be the most preferred habitat for most of the sand fly species since most of the collections (41.2%) were made from this site, followed by the rodent burrows.ConclusionsThe presence of P. orientalis and the high prevalence of VL in infants in the Al-Malha area provide the first evidence for local transmission of the parasite causing kala-azar in Darfur. Transmission is probably occurring during summer near the woodland where a high density of the vector was recorded. As a pre-requisite for designing effective control of VL in North Darfur, large scale entomological and epidemiological studies are recommended.

Faune phlébotomienne du foyer leishmanien de Keur Moussa : données actuelles sur la diversité et variations saisonnières (Thiès, Sénégal)

An inter-epidemic oversight was conducted in the cutaneous leishmaniasis focus of Keur Moussa (Thies region) between June 2015 and October 2016, more than 20 years after the last epidemic. The three sampling methods (adhesive traps, CDC light traps, and indoor pyrethroids sprays) allowed the capture of 1,746 sand flies belonging to 2 genera and 24 species, those involved in the transmission of leishmaniasis in Senegal, as well as 11 new species for the focus. The vector of human cutaneous leishmaniasis in Senegal, Phlebotomus duboscqi Neveu-Lemaire, 1906, represents 10.9% of this fauna. Sergentomyia schwetzi, one of the species involved, with Sergentomyia dubia and Sergentomyia magna, in the transmission of canine leishmaniasis in Senegal, is the most abundant species with 38.1% of the samples. The other two species have individually smaller percentages. Seasonal variations of the abundance show an intense activity of sandflies at the end of the dry season under the influence of high average temperatures and a humidity exceeding 50%. Rains are a limiting factor. According to the enrichment of the fauna and the high density of the different vectors of leishmaniasis in this focus, particularly P. duboscqi, a specific importance should be given for this focus in order to prevent occurrence of epidemics.

The mycobiota of the sand fly Phlebotomus perniciosus: Involvement of yeast symbionts in uric acid metabolism.

The knowledge of the fungal mycobiota of arthropods, including the vectors of human and animal diseases, is still limited. Here, the mycobiota associated with the sand fly Phlebotomus perniciosus, the main vector of leishmaniasis in the western Mediterranean area, by a culture-dependent approach (microbiological analyses and sequencing of the 26S rRNA gene), internal transcribed spacer (ITS) rRNA amplicon-based next-generation sequencing, fluorescence in situ hybridisation (FISH), and genome sequencing of the dominant yeast species was investigated. The dominant species was Meyerozyma guilliermondii, known for its biotechnological applications. The focus was on this yeast and its prevalence in adults, pupae and larvae of reared sand flies (overall prevalence: 57.5%) and of field-collected individuals (overall prevalence: 9%) was investigated. Using whole-mount FISH and microscopic examination, it was further showed that M. guilliermondii colonizes the midgut of females, males and larvae and the distal part of Malpighian tubules of female sand flies, suggesting a possible role in urate degradation. Finally, the sequencing and analysis of the genome of M. guilliermondii allowed predicting the complete uric acid degradation pathway, suggesting that the yeast could contribute to the removal of the excess of nitrogenous wastes after the blood meal of the insect host.

Ground ultra low volume (ULV) space spray applications for the control of wild sand fly populations (Psychodidae: Phlebotominae) in Europe

The Phlebotomus sand flies are considered an important vector of both canine and human leishmaniasis. Current measures for sand fly control include mostly indoor interventions, such as residual spraying of dwellings (IRS) to target endophilic sand fly species with very limited number of vector control tools for outdoor interventions against exophilic sand flies. In this study we investigated the efficacy of ground ultra low volume (ULV) space spray applications of a deltamethrin based product against field populations of P. perfiliewi, a major nuisance and pathogen-transmitting sand fly species of the Mediterranean Basin. Sand fly flight activity patterns and flight height preference within candidate treatment sites (kennels) were determined prior to treatments in order to optimize the timing and application parameters of the spray applications. On average there was a distinct activity peak between 20.00–22.00 h for both male and female P. perfiliewi with more than 45% and 30% of the population sampled occurring between 20.00–21.00 h and 21.00–22.00 h, respectively. No significant difference was observed in sand fly numbers from sticky traps placed at 0.5 up to 1.5 m height. However, there was a significant decrease in sand fly numbers at 2 m indicating a preference of sand flies to fly below 2 m. The low and high application rate of deltamethrin resulted in mean sand fly population decrease of 18 and 66%, respectively between pre-and post-treatment trap nights. The percent mean population change in the untreated control area was a positive number (30%) indicating that there was an increase in numbers of sand flies trapped between pre- and post-treatment nights. The results of this study provide strong evidence that ground ULV space spray applications when applied properly can result in significant sand fly control levels, even in a heavily infested sand fly environment such as the kennel sites used in this study.

Alternative splicing originates different domain structure organization of Lutzomyia longipalpis chitinases

BACKGROUNDThe insect chitinase gene family is composed by more than 10 paralogs, which can codify proteins with different domain structures. In Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil, a chitinase cDNA from adult female insects was previously characterized. The predicted protein contains one catalytic domain and one chitin-binding domain (CBD). The expression of this gene coincided with the end of blood digestion indicating a putative role in peritrophic matrix degradation.OBJECTIVESTo determine the occurrence of alternative splicing in chitinases of L. longipalpis.METHODSWe sequenced the LlChit1 gene from a genomic clone and the three spliced forms obtained by reverse transcription polymerase chain reaction (RT-PCR) using larvae cDNA.FINDINGSWe showed that LlChit1 from L. longipalpis immature forms undergoes alternative splicing. The spliced form corresponding to the adult cDNA was named LlChit1A and the two larvae specific transcripts were named LlChit1B and LlChit1C. The B and C forms possess stop codons interrupting the translation of the CBD. The A form is present in adult females post blood meal, L4 larvae and pre-pupae, while the other two forms are present only in L4 larvae and disappear just before pupation. Two bands of the expected size were identified by Western blot only in L4 larvae.MAIN CONCLUSIONSWe show for the first time alternative splicing generating chitinases with different domain structures increasing our understanding on the finely regulated digestion physiology and shedding light on a potential target for controlling L. longipalpis larval development.

Geospatial and Climatic Patterns Associated With the Distribution of Sand Fly (Diptera: Psychodidae) Vectors of Leishmaniasis in Pakistan.

We provide a comprehensive and updated review on the effects of elevation and climatic factors on distribution of sand fly (Diptera: Psychodidae) vectors of cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) across Pakistan. Our study was undertaken in view of the increasing risk of leishmaniasis, particularly resulting from climatic deviations and political instability in the region. Literature (published and unpublished) on suspected vectors of CL (Phlebotomus sergenti Parrot and Phlebotomus papatasi Scopoli) and VL (Phlebotomus major Yakimoff and Schokhor and Phlebotomus hindustanicus Theodor) was collected, sorted, and utilized in geospatial analysis. P. papatasi and P. sergenti co-occurred in majority of the referenced localities and were generally distributed toward the western and southern parts across a wide range of elevation, whereas P. major and P. hindustanicus were limited to high elevation in cool and humid northern areas of Pakistan. Conversely P. papatasi and P. sergenti exploited diverse ecological settings across the country, being particularly abundant in warm/hot and arid regions.

Ecology, feeding and natural infection by Leishmania spp. of phlebotomine sand flies in an area of high incidence of American tegumentary leishmaniasis in the municipality of Rio Branco, Acre, Brazil

BackgroundPhlebotomine sand flies (Diptera: Psychodidae) are insects of medical importance due to their involvement in the zoonotic transmission of Leishmania spp. to vertebrates. The aim of this work was to study the ecology of the sand fly fauna of two types of environments, a rural environment (the Transacreana Road) and an urban park (Horto Florestal Park), both located in the municipality of Rio Branco in the state of Acre, Brazil. Additionally, this study intended to investigate Leishmania infection and blood meal sources of these sand flies using molecular techniques.MethodsThe sand fly fauna was studied in different environments (i.e. forest and peridomestic environments in a rural area, and an urban forest) using Shannon traps and HP light traps to collect sand fly specimens over 13 consecutive months (December 2014 to January 2016). For investigating natural infection by Leishmania and the source of sand fly blood meals, DNA samples were extracted from female sand flies and subjected to polymerase chain reaction targeting ITS1 and cytb genes. DNA sequencing was subsequently used to identify species of Leishmania and the source of blood meals.ResultsA total of 2515 individual sand flies of 43 species were collected and identified, Trichophoromyia auraensis (839; 33.35%), Trichophoromyia spp. (537; 21.35%) and Evandromyia saulensis (187; 7.43%) were more abundant in the rural area (S = 41 species) than in the urban forest. No significant differences were found in species richness between forest and peridomestic environments in the rural area (H = 0.04; P > 0.05), but a larger number of species was found in the forest. Leishmania DNA was sequenced in 13 samples, confirming the presence of L. (V.) braziliensis in Th. auraensis (n = 1), Ev. saulensis (n = 2), Ev. walkeri (n = 1), Ps. llanosmartinsi (n = 1), Pi. nevesi (n = 2), Ps. davisi (n = 1), Ps. ayrozai (n = 1), Pa. aragaoi (n = 1), Ny. antunesi (n = 1) and Ev. infraspinosa (n = 1). Only Ps. ayrozai possessed a sequence similar to that of L. (V.) guyanensis (99%). Through microscopic analysis, five specimens of Ev. saulensis were found to possess flagellate forms in the hindgut, with an infection rate of 2.4%. Samples from 33 fed females were submitted to cytb gene amplification, for which sequencing determined that all were similar to the sequence deposited on GenBank for Gallus gallus (domestic chicken).ConclusionsThe high abundance of Trichophoromyia auraensis and Ev. saulensis, and the detection of L. (V.) braziliensis DNA, suggests that both species may be vectors of American tegumentary leishmaniasis. Psychodopygus ayrozai was found to be infected by L. (V) braziliesnsis and L. (V.) guyanensis, and although collected in low abundance, it may be a potential vector in the region. The sand fly fauna was found to be rich and diverse with predominance of the genus Psychodopygus. Identification of food sources of fed females showed that 100% amplified a gene region compatible with the domestic chicken, which although considered refractory in the disease transmission cycle, may have an influence on the population dynamics of sand flies.