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The present work assessed the experimental susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi. A L. (Leishmania) chagasi–Lu. longipalpis combination was used as a susceptible control. Wild-caught Ny. antunesi and laboratory-bred Lu. longipalpis were membrane-fed on blood with a 5 × 106/mL log-phase promastigote culture suspension and dissected on days 2 and 8 post-blood meal (pbm) for analysis focused on the assessment of parasitoses, as well as placement and promastigote morphotyping. Survival curves were constructed. In all combinations, promastigotes were observed on day 8 pbm. For both Leishmania species, in Lu. longipalpis, the presence of parasites was observed up to the stomodeal valve, while in Ny. antunesi, the presence of parasites was observed up to the cardia. There were no significant differences in parasitosis between L. (V.) lainsoni and L. (V.) lindenbergi in either Ny. antunesi or Lu. longipalpis. Six morphological promastigote forms were distinguished in Giemsa-stained gut smears. The survival curves of all combinations decreased and were affected differently by several Lu. longipalpis–parasite combinations, as well with Lu. longipalpis–uninfected blood. These findings stress Lu. longipalpis as experimentally susceptible to Leishmania spp. and suggest the putative susceptibility of Ny. antunesi to L. (V.) lainsoni and L. (V.) lindenbergi. [ABSTRACT FROM AUTHOR]

Sandflies are known vectors of leishmaniasis. In the Old World, sandflies are also vectors of viruses while little is known about the capacity of New World insects to transmit viruses to humans. Here, we relate the identification of RNA sequences with homology to rhabdovirus nucleocapsids (NcPs) genes, initially in the Lutzomyia longipalpis LL5 cell lineage, named NcP1.1 and NcP2. The Rhabdoviridae family never retrotranscribes its RNA genome to DNA. The sequences here described were identified in cDNA and DNA from LL-5 cells and in adult insects indicating that they are transcribed endogenous viral elements (EVEs). The presence of NcP1.1 and NcP2 in the L. longipalpis genome was confirmed in silico. In addition to showing the genomic location of NcP1.1 and NcP2, we identified another rhabdoviral insertion named NcP1.2. Analysis of small RNA molecules derived from these sequences showed that NcP1.1 and NcP1.2 present a profile consistent with elements targeted by primary piRNAs, while NcP2 was restricted to the degradation profile. The presence of NcP1.1 and NcP2 was investigated in sandfly populations from South America and the Old World. These EVEs are shared by different sandfly populations in South America while none of the Old World species studied presented the insertions. [ABSTRACT FROM AUTHOR]

Abstract Background Understanding aspects related to the physiology and capacity of vectors is essential for effectively controlling vector-borne diseases. The sand fly Lutzomyia longipalpis has great importance in medical entomology for disseminating Leishmania parasites, the causative agent of Leishmaniasis, one of the main neglected diseases listed by the World Health Organization (WHO). In this respect, it is necessary to evaluate the transmission potential of this species and the success of vector control interventions. Near-infrared spectroscopy (NIRS) has been used to estimate the age of mosquitoes in different conditions (laboratory, semi-field, and conservation), taxonomic analysis, and infection detection. However, no studies are using NIRS for sand flies. Methods In this study, we developed analytic models to estimate the age of L. longipalpis adults under laboratory conditions, identify their copulation state, and evaluate their gonotrophic cycle and diet. Results Sand flies were classified with an accuracy of 58–82% in 3 age groups and 82–92% when separating them into young (8 days) insects. The classification between mated and non-mated sandflies was 98–100% accurate, while the percentage of hits of females that had already passed the first gonotrophic cycle was only 59%. Conclusions We consider the age and copula estimation results very promising, as they provide essential aspects of vector capacity assessment, which can be obtained quickly and at a lower cost with NIRS. Graphical Abstract

Background: Understanding aspects related to the physiology and capacity of vectors is essential for effectively controlling vector-borne diseases. The sand fly Lutzomyia longipalpis has great importance in medical entomology for disseminating Leishmania parasites, the causative agent of Leishmaniasis, one of the main neglected diseases listed by the World Health Organization (WHO). In this respect, it is necessary to evaluate the transmission potential of this species and the success of vector control interventions. Near-infrared spectroscopy (NIRS) has been used to estimate the age of mosquitoes in different conditions (laboratory, semi-field, and conservation), taxonomic analysis, and infection detection. However, no studies are using NIRS for sand flies. Methods: In this study, we developed analytic models to estimate the age of L. longipalpis adults under laboratory conditions, identify their copulation state, and evaluate their gonotrophic cycle and diet. Results: Sand flies were classified with an accuracy of 58–82% in 3 age groups and 82–92% when separating them into young (<8 days) or old (>8 days) insects. The classification between mated and non-mated sandflies was 98–100% accurate, while the percentage of hits of females that had already passed the first gonotrophic cycle was only 59%. Conclusions: We consider the age and copula estimation results very promising, as they provide essential aspects of vector capacity assessment, which can be obtained quickly and at a lower cost with NIRS. Key message: Determining adult dipterans sand flies' age and mating status may be necessary for critical assessment of vector control interventions. Near-infrared spectroscopy (NIRS) is a fast, low-cost technique with many advantages compared to the traditional techniques that follow ovarian or cuticle development or pteridine content. In this work, we show for the first time to our knowledge that NIRS may be used for discrimination between young and old sand flies and mated or non-mated adults of both sexes in the Leishmania vector Lutzomyia longipalpis. [ABSTRACT FROM AUTHOR]

Background: The successful use of semiochemicals to attract insects to traps is based on research on the most suitable compounds and their release profiles over time. Based on the group's promising results, matrices with a more adequate release profile and more eco-friendly properties for the release of 1-hexanol were developed. To use a more suitable prototype in the field, the most promising systems were added to a capsule and evaluated in a wind tunnel. Behavioral experiments were performed using the sand fly species, Lutzomyia longipalpis, to evaluate the efficacy of the proposed system. Methods: Different delivery systems were developed by varying the polymer (gellan gum and pectin) ratio, crosslinker (aluminum chloride) concentration, and glutaraldehyde removal.The delivery systems were loaded with 1-hexanol, and their release profiles were evaluated using gravimetric analysis under ambient and high-humidity conditions. When the matrix system was placed inside a plastic container, modulations in the active release profile were observed and the system could be reused. Actid attraction behaviors of the sand fly species, Lu. longipalpis, were evaluated in a wind tunnel when exposed to 1-hexanol-loaded release systems at different times. Results: Among the four formulations evaluated, System 2 (gellan gum and pectin in a 1:1 ratio with 5% aluminum chloride) exhibited the most promising release profile, with greater uniformity and longer compound release time. The maximum 1-hexanol release uniformity was achieved over a longer time, mainly every 24 h, under both ambient and high-humidity conditions. System 2 can be reused at least once with the same structure. The wind tunnel trials exhibited efficient activation and attraction of Lu. longipalpis to 1-hexanol after 24, 48, and 72 h in System 2 placed inside the capsules. Conclusions: The polymeric matrix supplemented with 1-hexanol and introduced in plastic capsules showed promising results in attracting sand flies. This system can be used as a solution for other attractive compounds as well as in other applications where their release needs to be controlled or prolonged. [ABSTRACT FROM AUTHOR]

Abstract Background The successful use of semiochemicals to attract insects to traps is based on research on the most suitable compounds and their release profiles over time. Based on the group's promising results, matrices with a more adequate release profile and more eco-friendly properties for the release of 1-hexanol were developed. To use a more suitable prototype in the field, the most promising systems were added to a capsule and evaluated in a wind tunnel. Behavioral experiments were performed using the sand fly species, Lutzomyia longipalpis, to evaluate the efficacy of the proposed system. Methods Different delivery systems were developed by varying the polymer (gellan gum and pectin) ratio, crosslinker (aluminum chloride) concentration, and glutaraldehyde removal.The delivery systems were loaded with 1-hexanol, and their release profiles were evaluated using gravimetric analysis under ambient and high-humidity conditions. When the matrix system was placed inside a plastic container, modulations in the active release profile were observed and the system could be reused. Actid attraction behaviors of the sand fly species, Lu. longipalpis, were evaluated in a wind tunnel when exposed to 1-hexanol-loaded release systems at different times. Results Among the four formulations evaluated, System 2 (gellan gum and pectin in a 1:1 ratio with 5% aluminum chloride) exhibited the most promising release profile, with greater uniformity and longer compound release time. The maximum 1-hexanol release uniformity was achieved over a longer time, mainly every 24 h, under both ambient and high-humidity conditions. System 2 can be reused at least once with the same structure. The wind tunnel trials exhibited efficient activation and attraction of Lu. longipalpis to 1-hexanol after 24, 48, and 72 h in System 2 placed inside the capsules. Conclusions The polymeric matrix supplemented with 1-hexanol and introduced in plastic capsules showed promising results in attracting sand flies. This system can be used as a solution for other attractive compounds as well as in other applications where their release needs to be controlled or prolonged. Graphical Abstract

Background: Porteirinha is endemic for visceral leishmaniasis (VL), with intense disease transmission of the disease. We evaluated the impact of canine euthanasia as a single control measure on the incidence of VL in humans and canines. Methods: A prospective observational cohort study was carried out over four years (1998–2002) in 8 of the 12 neighborhoods of the city. The dynamics of canine visceral leishmaniasis (CVL) transmission were evaluated for 2 years, before beginning the screening–culling intervention. The comparative morbidity index (CMI) was used to stratify areas with the greatest risk of CVL, and the spatial distribution of human and canine VL cases was compared using univariate and bivariate K-functions. Results: Human cases conglomerated in three neighborhoods. Spatial clusters were detected for CVL in 1998, 2000, and 2001, but not in 1999, when greater spatial dispersion occurred. The screening and culling intervention reduced the number of human VL cases and decreased the incidence of CVL, mainly in neighborhoods with a high CMI. Conclusions: The systematic euthanasia of seropositive dogs was shown to be an effective control action of the Program for Control of Visceral Leishmaniasis (PCLV) in Brazil. The fundamental role of domestic dogs in the epidemiological chain of VL was reaffirmed. [ABSTRACT FROM AUTHOR]

The present work assessed the experimental susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi. A L. (Leishmania) chagasi–Lu. longipalpis combination was used as a susceptible control. Wild-caught Ny. antunesi and laboratory-bred Lu. longipalpis were membrane-fed on blood with a 5 × 106/mL log-phase promastigote culture suspension and dissected on days 2 and 8 post-blood meal (pbm) for analysis focused on the assessment of parasitoses, as well as placement and promastigote morphotyping. Survival curves were constructed. In all combinations, promastigotes were observed on day 8 pbm. For both Leishmania species, in Lu. longipalpis, the presence of parasites was observed up to the stomodeal valve, while in Ny. antunesi, the presence of parasites was observed up to the cardia. There were no significant differences in parasitosis between L. (V.) lainsoni and L. (V.) lindenbergi in either Ny. antunesi or Lu. longipalpis. Six morphological promastigote forms were distinguished in Giemsa-stained gut smears. The survival curves of all combinations decreased and were affected differently by several Lu. longipalpis–parasite combinations, as well with Lu. longipalpis–uninfected blood. These findings stress Lu. longipalpis as experimentally susceptible to Leishmania spp. and suggest the putative susceptibility of Ny. antunesi to L. (V.) lainsoni and L. (V.) lindenbergi.

Sandflies are known vectors of leishmaniasis. In the Old World, sandflies are also vectors of viruses while little is known about the capacity of New World insects to transmit viruses to humans. Here, we relate the identification of RNA sequences with homology to rhabdovirus nucleocapsids (NcPs) genes, initially in the Lutzomyia longipalpis LL5 cell lineage, named NcP1.1 and NcP2. The Rhabdoviridae family never retrotranscribes its RNA genome to DNA. The sequences here described were identified in cDNA and DNA from LL-5 cells and in adult insects indicating that they are transcribed endogenous viral elements (EVEs). The presence of NcP1.1 and NcP2 in the L. longipalpis genome was confirmed in silico. In addition to showing the genomic location of NcP1.1 and NcP2, we identified another rhabdoviral insertion named NcP1.2. Analysis of small RNA molecules derived from these sequences showed that NcP1.1 and NcP1.2 present a profile consistent with elements targeted by primary piRNAs, while NcP2 was restricted to the degradation profile. The presence of NcP1.1 and NcP2 was investigated in sandfly populations from South America and the Old World. These EVEs are shared by different sandfly populations in South America while none of the Old World species studied presented the insertions.

ABSTRACT Background: Here, Leishmania presence in sand flies from Três Lagoas, Mato Grosso do Sul, Brazil, after visceral leishmaniasis (VL) was investigated. Methods: In April 2022, two light traps were deployed within and around the residence for two days post-VL case report. Results: A total of 120 Lutzomyia longipalpis were collected. Suprapyloric flagellates were found in a female sand fly with eggs and residual blood during midgut dissection. Sequencing of ITS1 and cytb fragments confirmed Leishmania infantum DNA and identified Homo sapiens as the blood source, respectively. Conclusions: This study emphasizes the importance of monitoring sand flies in VL endemic areas.

Lutzomyia longipalpis is known as one of the primary insect vectors of visceral leishmaniasis. For such ectothermic organisms, the ambient temperature is a critical life factor. However, the impact of temperature has been ignored in many induced‐stress situations of the vector life. Therefore, this study explored the interaction of Lu. longipalpis with temperature by evaluating its behaviour across a thermal gradient, thermographic recordings during blood‐feeding on mice, and the gene expression of heat shock proteins (HSP) when insects were exposed to extreme temperature or infected. The results showed that 72 h after blood ingestion, Lu. longipalpis became less active and preferred relatively low temperatures. However, at later stages of blood digestion, females increased their activity and remained at higher temperatures. Real‐time imaging showed that the body temperature of females can adjust rapidly to the host and remain constant until the end of blood‐feeding. Insects also increased the expression of HSP90(83) during blood‐feeding. Our findings suggest that Lu. longipalpis interacts with temperature by using its behaviour to avoid temperature‐induced physiological damage during the gonotrophic cycle. However, the expression of certain HSP might be triggered to mitigate thermal stress in situations where a behavioural response is not the best option. [ABSTRACT FROM AUTHOR]

Background:Bartonella ancashensis is a recently described Bartonella species endemic to Peru, where it causes verruga peruana in humans. While the arthropod vector of B. ancashensis transmission is unknown, human coinfections with Bartonella bacilliformis suggest that phlebotomine sand flies are a vector. Materials and Methods: To address the hypothesis that sand flies are involved in the bacterium's transmission, Lutzomyia longipalpis sand flies were used as an infection model, together with green fluorescent protein-expressing B. ancashensis. Results: Results showed that bacterial infections were clearly established, limited to the anterior midgut of the female fly, and maintained for roughly 7 days. At 3–7 days postinfection, a prominent microcolony of aggregated bacteria was observed in the anterior midgut, immediately distal to the stomodeal valve of the esophagus. In contrast, eggs, diuretic fluid, feces, and other tissues were not infected. Conclusion: These results suggest that certain sand fly species within the endemic zone for B. ancashensis may play a role in the bacterium's maintenance and possibly in its transmission to humans. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis remains a serious public health issue, and Brazil was among the seven countries with the highest prevalence of this disease worldwide. The measures to control this disease are not easily developed, and the improvement of its diagnosis, surveillance, and control is still needed. This study aimed to carry out the polymerase chain reaction (PCR) diagnosis of Leishmania infantum in vector samples in some municipalities of the State of São Paulo, which included two municipalities with human disease transmission and two with dog transmission only. Vectors were collected in traps with luminous bait. Next, they were killed at −4 °C and kept in 70% alcohol. Groups of ten female insects (pools) were mashed on cation exchange paper (fine cellulose phosphate with 18 µEq/cm² ionic exchange capacity) for DNA extraction. The PCR was carried out to identify the natural infection of the Leishmania genus in female Lutzomyia longipalpis (Lu. Longipalpis). Out of the 3,880 Lu. longipalpis phlebotomines, 1060 were female and 2820 were male (3:1). The method used to extract the DNA in pools of ten phlebotomines and the PCR resulted in sensitivity, specificity, practicality, and faster analyses when compared to the individual analysis method. The procedure described can be used on a large scale in the leishmaniasis epidemiological surveillance, enabling a higher number of analyses and the optimization of human resources because the traditional diagnostic method is carried out via desiccation of the insect digestive system and microscopic examination, which is time-demanding and there is the need of manual skills. [ABSTRACT FROM AUTHOR]

Background: Porteirinha is endemic for visceral leishmaniasis (VL), with intense disease transmission of the disease. We evaluated the impact of canine euthanasia as a single control measure on the incidence of VL in humans and canines. Methods: A prospective observational cohort study was carried out over four years (1998–2002) in 8 of the 12 neighborhoods of the city. The dynamics of canine visceral leishmaniasis (CVL) transmission were evaluated for 2 years, before beginning the screening–culling intervention. The comparative morbidity index (CMI) was used to stratify areas with the greatest risk of CVL, and the spatial distribution of human and canine VL cases was compared using univariate and bivariate K-functions. Results: Human cases conglomerated in three neighborhoods. Spatial clusters were detected for CVL in 1998, 2000, and 2001, but not in 1999, when greater spatial dispersion occurred. The screening and culling intervention reduced the number of human VL cases and decreased the incidence of CVL, mainly in neighborhoods with a high CMI. Conclusions: The systematic euthanasia of seropositive dogs was shown to be an effective control action of the Program for Control of Visceral Leishmaniasis (PCLV) in Brazil. The fundamental role of domestic dogs in the epidemiological chain of VL was reaffirmed.

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. [ABSTRACT FROM AUTHOR]

Leishmaniasis control often relies upon insecticidal control of phlebotomine sandfly vector populations. Such methods are vulnerable to the evolution of insecticide resistance via a range of molecular mechanisms. There is evidence that two major resistance mechanisms, target site insensitivity and metabolic resistance, have evolved in some sandfly populations and further genetic characterization of resistance would be useful to understand and combat it. To facilitate the study of the mechanisms of metabolic resistance, here we improved the annotation and characterized a major detoxification gene family, the glutathione‐s‐transferases (GST), in the genomes of two sand fly species: Phlebotomus papatasi and Lutzomyia longipalpis. The compositions of the GST gene family differ markedly from those of Aedes and Anopheles mosquitoes. Most strikingly, the xi (X) class of GSTs appears to have expanded in both sand fly genomes. Our results provide a basis for further studies of metabolic resistance mechanisms in these important disease vector species. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected tropical disease that continues to pose a serious public health problem. Albeit dogs have long been held as the major reservoirs of Leishmania infantum, the involvement of domestic cats in the zoonotic cycle of visceral leishmaniasis has gained prominence. Here, 240 cats were evaluated by clinical signs and haematological/biochemical changes compatible with leishmaniasis and were diagnosed by serological, molecular, and parasitological techniques. Thus, four cats naturally infected by L. infantum were submitted to xenodiagnosis. A total of 203 females of Lutzomyia longipalpis were subjected to feeding on four cats, with all females completing the blood meal. Parasitological and molecular assays were carried out to evaluate the presence of L. infantum in the sand flies' midgut. Promastigotes were observed in 10 females (6.5%) that fed on one cat, and L. infantum DNA was detected in 17 (8.4%) females that fed on two cats. Our results strengthen the evidence that naturally infected cats are capable of transmitting L. infantum to sand flies. [ABSTRACT FROM AUTHOR]

Abstract Insecticides can exert strong selection on insect pest species, including those that vector diseases, and have led to rapid evolution of resistance. Despite such rapid evolution, relatively little is known about standing genetic variation for resistance in insecticide‐susceptible populations of many species. To help fill this knowledge gap, we generated genotyping‐by‐sequencing data from insecticide‐susceptible Phlebotomus papatasi and Lutzomyia longipalpis sand flies that survived or died from a sub‐diagnostic exposure to either permethrin or malathion using a modified version of the Centers for Disease Control and Prevention bottle bioassay. Multi‐locus genome‐wide association mapping methods were used to quantify standing genetic variation for insecticide resistance in these populations and to identify specific alleles associated with insecticide survival. For each insecticide treatment, we estimated the proportion of the variation in survival explained by the genetic data (i.e., “chip” heritability) and the number and contribution of individual loci with measurable effects. For all treatments, survival to an insecticide exposure was heritable with a polygenic architecture. Both P. papatasi and L. longipalpis had alleles for survival that resided within many genes throughout their genomes. The implications for resistance conferred by many alleles, as well as inferences made about the utility of laboratory insecticide resistance association studies compared to field observations, are discussed.

Visceral leishmaniasis (VL) is a neglected tropical disease transmitted by Lutzomyia longipalpis, a sand fly widely distributed in Brazil. Despite efforts to strengthen national control programs reduction in incidence and geographical distribution of VL in Brazil has not yet been successful; VL is in fact expanding its range in newly urbanized areas. Ecological niche models (ENM) for use in surveillance and response systems may enable more effective operational VL control by mapping risk areas and elucidation of eco-epidemiologic risk factors. ENMs for VL and Lu. longipalpis were generated using monthly WorldClim 2.0 data (30-year climate normal, 1-km spatial resolution) and monthly soil moisture active passive (SMAP) satellite L4 soil moisture data. SMAP L4 Global 3-hourly 9-km EASE-Grid Surface and Root Zone Soil Moisture Geophysical Data V004 were obtained for the first image of day 1 and day 15 (0:00-3:00 hour) of each month. ENM were developed using MaxEnt software to generate risk maps based on an algorithm for maximum entropy. The jack-knife procedure was used to identify the contribution of each variable to model performance. The three most meaningful components were used to generate ENM distribution maps by ArcGIS 10.6. Similar patterns of VL and vector distribution were observed using SMAP as compared to WorldClim 2.0 models based on temperature and precipitation data or water budget. Results indicate that direct Earth-observing satellite measurement of soil moisture by SMAP can be used in lieu of models calculated from classical temperature and precipitation climate station data to assess VL risk.

Abstract Background Phlebotomines are a group of insects which include vectors of the Leishmania parasites that cause visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL), diseases primarily affecting populations of low socioeconomic status. VL in Brazil is caused by Leishmania infantum, with transmission mainly attributed to Lutzomyia longipalpis, a species complex of sand fly, and is concentrated mainly in the northeastern part of the country. CL is distributed worldwide and occurs in five regions of Brazil, at a higher incidence in the north and northeast regions, with etiological agents, vectors, reservoirs and epidemiological patterns that differ from VL. The aim of this study was to determine the composition, distribution and ecological relationships of phlebotomine species in an Atlantic Forest conservation unit and nearby residential area in northeastern Brazil. Methods Centers for Disease Control and Shannon traps were used for collections, the former at six points inside the forest and in the peridomestic environment of surrounding residences, three times per month for 36 months, and the latter in a forest area, once a month for 3 months. The phlebotomines identified were compared with climate data using simple linear correlation, Pearson’s correlation coefficient and cross-correlation. The estimate of ecological parameters was calculated according to the Shannon-Wiener diversity index, standardized index of species abundance and the dominance index. Results A total of 75,499 phlebotomines belonging to 11 species were captured in the CDC traps, the most abundant being Evandromyia walkeri, Psychodopygus wellcomei and Lu. longipalpis. Evandromyia walkeri abundance was most influenced by temperature at collection time and during the months preceding collection and rainfall during the months preceding collection. Psychodopygus wellcomei abundance was most affected by rainfall and relative humidity during the collection month and the month immediately preceding collection time. Lutzomyia longipalpis abundance showed a correlation with temperature and the rainfall during the months preceding collection time. The Shannon trap contained a total of 3914 phlebotomines from these different species. Psychodopygus wellcomei, accounting for 91.93% of the total, was anthropophilic and active mainly at night. Conclusions Most of the species collected in the traps were seasonal and exhibited changes in their composition and population dynamics associated with local adaptions. The presence of vectors Ps. wellcomei and Lu. longipalpis underscore the epidemiological importance of these phlebotomines in the conservation unit and surrounding anthropized areas. Neighboring residential areas should be permanently monitored to prevent VL or CL transmission and outbreaks.

Abstract Background Leishmania infantum is the etiological agent of visceral leishmaniasis (VL) in the New World, where the sand fly Lutzomyia longipalpis and domestic dogs are considered the main vector and host reservoirs, respectively. Systemic insecticides have been studied as an alternative to control vector-borne diseases, including VL. Fluralaner, an isoxazoline class compound, is a systemic insecticide used in dogs, with proven efficiency against different species of phlebotomine sand flies. However, to date no studies have demonstrated the efficacy of fluralaner on Lu. longipalpis. The aim of this study was to evaluate the insecticidal effect of fluralaner (Bravecto®) on the sand fly Lu. longipalpis after blood meal in treated dogs. Methods Healthy mongrel dogs (n = 8) were recruited from the Zoonoses Control Center in the city of Natal, Rio Grande do Norte, Brazil, and randomized into two groups: fluralaner treated (n = 4) and non-treated control (n = 4). Colony-reared female specimens of Lu. longipalpis (n = 20) were allowed to feed on all dogs for 40 min before treatment (for fluralaner-treated dogs), at day 1 after treatment and then monthly until 1 year post-treatment. Results In the treatment group, there was 100% mortality of Lu. longipalpis for up to 5 months after treatment initiation, decreasing to 72.5% at 6 months post-treatment initiation. The efficacy of fluralaner ranged from 100% at day 1 (P = 0.0002) to 68% ( P = 0.0015) at 6 months, decreasing to 1.4% at 1 year post-treatment. Sand fly mortality carried out blood meal in non-treated control dogs remained constant at ≤ 15%. Conclusions Taken together, our results suggest that fluralaner may be used as a control strategy for VL in dogs in VL endemic areas.

Abstract Background In South America, cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) are emerging diseases, expanding in the border area of Argentina, Brazil and Paraguay. Outbreaks of CL were reported since the 1990s, with Nyssomyia whitmani as the main vector in this region. Regarding VL, urban reports started in 2010 with Lutzomyia longipalpis as the main vector. The aim of this study was to evaluate environmental determinants related to the main vectors of leishmaniasis, to contribute to the prevention and control response to the emergence of VL and CL in the Argentina-Brazil-Paraguay border region. Methods The cross-sectional survey includes two cities and two close rural areas in the Argentinean Northeast Region, between November 2014 and January 2015, with a total of 95 sampling sites. REDILA-BL traps were set for three consecutive nights, and a total of 68 meso- and microscale environmental and landscape characteristics were surveyed. The association between vector abundance with different variables was evaluated using a generalized linear model with zero-inflated negative binomial distribution. We analyzed females for detection of Leishmania DNA. Results The analysis for Lu. longipalpis indicates an excess of absences when the mean NDWI around the sites were higher. The abundance of Lu. longipalpis at mesoscale level was higher when more urban services were present, and when blood sources such as chickens or dogs at the microscale level were present. For Ny. whitmani, no variable was found to be associated with the absences, while its abundance increased in association with the following variables: percentage of tree cover, presence of garbage collection service, hosted people and, at microscale, the presence of poultry. Leshmania infantum DNA was detected in 2/49 (4%) Lu. longipalpis. Conclusions The abundance of both species is influenced by variables at different scales, their influence probably has a hierarchy and they are acting on different aspects of the biology of these vectors. The urban spatial segregation of Lu. longipalpis and the peri-urban and rural segregation of N. whitmani increase the risk of VL and CL. The selection of the better variables for each scale will allow the design of appropriate control strategies depending on species.

Abstract Background Sand flies are the vectors of Leishmania parasites. To develop in the sand fly midgut, Leishmania multiplies and undergoes various stage differentiations giving rise to the infective form, the metacyclic promastigotes. To determine the changes in sand fly midgut gene expression caused by the presence of Leishmania, we performed RNA-Seq of uninfected and Leishmania infantum-infected Lutzomyia longipalpis midguts from seven different libraries corresponding to time points which cover the various Leishmania developmental stages. Results The combined transcriptomes resulted in the de novo assembly of 13,841 sand fly midgut transcripts. Importantly, only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Further, we observed distinct differentially expressed sand fly midgut transcripts corresponding to the presence of each of the various Leishmania stages suggesting that each parasite stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1–4), more transcripts were down-regulated by Leishmania infection at large fold changes (> 32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes (

Objectives: This study evaluated the implications of clinically acquired miltefosine resistance (MIL-R) by assessing virulence in mice and sand flies to reveal the potential of MIL-R strains to circulate. Methods: Experimental infections with the MIL-R clinical Leishmania infantum isolate MHOM/FR/2005/LEM5159, having a defect in the LiROS3 subunit of the MIL-transporter, and its syngeneic experimentally reconstituted MIL-S counterpart (LEM5159LiROS3) were performed in BALB/c mice and Lutzomyia longipalpis and Phlebotomus perniciosus sand flies. In mice, the amastigote burdens in liver and spleen were compared microscopically using Giemsa smears and by bioluminescent imaging. During the sand fly infections, the percentage of infected flies, parasite load, colonization of the stomodeal valve and metacyclogenesis were evaluated. The stability of the MIL-R phenotype after sand fly and mouse passage was determined as well. Results: The fitness of the MIL-R strain differed between the mouse and sand fly infection model. In mice, a clear fitness loss was observed compared to the LiROS3-reconstituted susceptible strain. This defect could be rescued by episomal reconstitution with a wildtype LiROS3 copy. However, this fitness loss was not apparent in the sand fly vector, resulting in metacyclogenesis and efficient colonization of the stomodeal valve. Resistance was stable after passage in both sand fly and mouse. Conclusion: The natural MIL-R strain is significantly hampered in its ability to multiply and cause a typical visceral infection pattern in BALB/c mice. However, this LiROS3-deficient strain efficiently produced mature infections and metacyclic promastigotes in the sand fly vector highlighting the transmission potential of this particular MIL-R clinical Leishmania strain.

Abstract Background Since the introduction of miltefosine (MIL) as first-line therapy in the kala-azar elimination programme in the Indian subcontinent, treatment failure rates have been increasing. Since parasite infectivity and virulence may become altered upon treatment relapse, this laboratory study assessed the phenotypic effects of repeated in vitro and in vivo MIL exposure. Methods Syngeneic Leishmania donovani lines either or not exposed to MIL were compared for drug susceptibility, rate of promastigote multiplication and metacyclogenesis, macrophage infectivity and behaviour in the sand fly vector, Lutzomyia longipalpis. Results Promastigotes of both in vitro and in vivo MIL-selected strains displayed a slightly reduced drug susceptibility that was associated with a reduced MIL-accumulation linked to a lower copy number (disomic state) of chromosome 13 harboring the miltefosine transporter (LdMT) gene. In vitro selected promastigotes showed a lower rate of metacyclogenesis whereas the in vivo derived promastigotes displayed a moderately increased growth rate. Repeated MIL exposure did neither influence the parasite load nor metacyclogenesis in the sand fly vector. Conclusions Recurrent in vitro and in vivo MIL exposure evokes a number of very subtle phenotypic and genotypic changes which could make promastigotes less susceptible to MIL without attaining full resistance. These changes did not significantly impact on infection in the sand fly vector.

Visceral leishmaniasis remains a serious public health issue, and Brazil was among the seven countries with the highest prevalence of this disease worldwide. The measures to control this disease are not easily developed, and the improvement of its diagnosis, surveillance, and control is still needed. This study aimed to carry out the polymerase chain reaction (PCR) diagnosis of Leishmania infantum in vector samples in some municipalities of the State of São Paulo, which included two municipalities with human disease transmission and two with dog transmission only. Vectors were collected in traps with luminous bait. Next, they were killed at −4 °C and kept in 70% alcohol. Groups of ten female insects (pools) were mashed on cation exchange paper (fine cellulose phosphate with 18 µEq/cm² ionic exchange capacity) for DNA extraction. The PCR was carried out to identify the natural infection of the Leishmania genus in female Lutzomyia longipalpis (Lu. Longipalpis). Out of the 3,880 Lu. longipalpis phlebotomines, 1060 were female and 2820 were male (3:1). The method used to extract the DNA in pools of ten phlebotomines and the PCR resulted in sensitivity, specificity, practicality, and faster analyses when compared to the individual analysis method. The procedure described can be used on a large scale in the leishmaniasis epidemiological surveillance, enabling a higher number of analyses and the optimization of human resources because the traditional diagnostic method is carried out via desiccation of the insect digestive system and microscopic examination, which is time-demanding and there is the need of manual skills.

Background: Phlebotomines are a group of insects which include vectors of the Leishmania parasites that cause visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL), diseases primarily affecting populations of low socioeconomic status. VL in Brazil is caused by Leishmania infantum, with transmission mainly attributed to Lutzomyia longipalpis, a species complex of sand fly, and is concentrated mainly in the northeastern part of the country. CL is distributed worldwide and occurs in five regions of Brazil, at a higher incidence in the north and northeast regions, with etiological agents, vectors, reservoirs and epidemiological patterns that differ from VL. The aim of this study was to determine the composition, distribution and ecological relationships of phlebotomine species in an Atlantic Forest conservation unit and nearby residential area in northeastern Brazil. Methods: Centers for Disease Control and Shannon traps were used for collections, the former at six points inside the forest and in the peridomestic environment of surrounding residences, three times per month for 36 months, and the latter in a forest area, once a month for 3 months. The phlebotomines identified were compared with climate data using simple linear correlation, Pearson's correlation coefficient and cross-correlation. The estimate of ecological parameters was calculated according to the Shannon-Wiener diversity index, standardized index of species abundance and the dominance index. Results: A total of 75,499 phlebotomines belonging to 11 species were captured in the CDC traps, the most abundant being Evandromyia walkeri, Psychodopygus wellcomei and Lu. longipalpis. Evandromyia walkeri abundance was most influenced by temperature at collection time and during the months preceding collection and rainfall during the months preceding collection. Psychodopygus wellcomei abundance was most affected by rainfall and relative humidity during the collection month and the month immediately preceding collection time. Lutzomyia longipalpis abundance showed a correlation with temperature and the rainfall during the months preceding collection time. The Shannon trap contained a total of 3914 phlebotomines from these different species. Psychodopygus wellcomei, accounting for 91.93% of the total, was anthropophilic and active mainly at night. Conclusions: Most of the species collected in the traps were seasonal and exhibited changes in their composition and population dynamics associated with local adaptions. The presence of vectors Ps. wellcomei and Lu. longipalpis underscore the epidemiological importance of these phlebotomines in the conservation unit and surrounding anthropized areas. Neighboring residential areas should be permanently monitored to prevent VL or CL transmission and outbreaks. [ABSTRACT FROM AUTHOR]

Vector-borne diseases (VBDs) are important contributors to the global disease burden and are a key factor in perpetuating economic inequality. Although environmental changes are often cited as drivers of VBDs, the link between deforestation and VBD occurrence remains unclear. Here, we examined this relationship in detail using the spread of visceral leishmaniasis (VL) in São Paulo state (Brazil) as the case study. We used a two-step approach to estimate the causal effects (overall, direct, and indirect) of deforestation on the occurrence of the VL vector, canine visceral leishmaniasis (CVL), and human visceral leishmaniasis (HVL). We first estimated the parameters via a double Metropolis–Hastings algorithm and then estimated the causal effects through a Gibbs sampler. We observed that the odds of vector, CVL, and HVL occurrence were 2.63-, 2.07-, and 3.18-fold higher, respectively, in deforested compared with forested municipalities. We also identified a significant influence of the presence of vector, CVL, and HVL in one municipality on disease occurrence in previously naive neighbouring municipalities. Lastly, we found that a hypothetical reduction in deforestation prevalence from 50 to 0% across the state would reduce vector, CVL, and HVL occurrence by 11%, 6.67%, and 29.87%, respectively. Our results suggest that implementing an eco-friendly development strategy that considers trade-offs between agriculture, urbanization, and conservation could be an effective mechanism of controlling VL. [ABSTRACT FROM AUTHOR]

In many animal species, sex determination is accomplished by heterogamety i.e., one of the sexes produces two types of gametes, which upon fertilization will direct the development toward males or females. Both male ("XY") and female ("ZW") heterogamety are known to occur and can be easily distinguished when the sex-chromosomes are morphologically different. However, this approach fails in cases of homomorphic sex chromosomes, such as the sand fly Lutzomyia longipalpis s.l. (Psychodidae, Diptera), which is the main vector of visceral leishmaniosis in Brazil. In order to identify the heterogametic sex in L. longipalpis s.l., we did a whole-genome sequencing of males and females separately and used the "Y chromosome Genome Scan" (YGS) method to find sex-specific sequences. Our results, which were confirmed by PCR, show that L. longipalpis s.l. has XY system. The YGS method can be especially useful in situations in which no morphological difference is observed in the sex-chromosomes or when fresh specimens are not readily available. [ABSTRACT FROM AUTHOR]

A male canine, two years old, with no known breed, with a history of diarrhea and ticks, was submitted to a private veterinary clinic in the City of Guarulhos, São Paulo State, Brazil: presenting weight loss, alopecia and intense pruritus. The owner reported that the dog was a native of the City of Votuporanga, São Paulo State. Blood samples were collected for blood counts and biochemical measurements, and rapid tests were performed with commercial kits for the detection of Ehrlichia canis, Anaplasma spp., Borrelia burgdorferi and Canine distemper. Due to the hematological and biochemical changes, the negative results, and the origin of the animal, it was decided to perform a rapid test for leishmaniasis, where this test presented a positive result, later confirmed by Polymerase Chain Reaction. The Leishmaniose continue advancing, being able to settle in non-endemic areas.

Insecticides can exert strong selection on insect pest species, including those that vector diseases, and have led to rapid evolution of resistance. Despite such rapid evolution, relatively little is known about standing genetic variation for resistance in insecticide‐susceptible populations of many species. To help fill this knowledge gap, we generated genotyping‐by‐sequencing data from insecticide‐susceptible Phlebotomus papatasi and Lutzomyia longipalpis sand flies that survived or died from a sub‐diagnostic exposure to either permethrin or malathion using a modified version of the Centers for Disease Control and Prevention bottle bioassay. Multi‐locus genome‐wide association mapping methods were used to quantify standing genetic variation for insecticide resistance in these populations and to identify specific alleles associated with insecticide survival. For each insecticide treatment, we estimated the proportion of the variation in survival explained by the genetic data (i.e., "chip" heritability) and the number and contribution of individual loci with measurable effects. For all treatments, survival to an insecticide exposure was heritable with a polygenic architecture. Both P. papatasi and L. longipalpis had alleles for survival that resided within many genes throughout their genomes. The implications for resistance conferred by many alleles, as well as inferences made about the utility of laboratory insecticide resistance association studies compared to field observations, are discussed. [ABSTRACT FROM AUTHOR]

Abstract Background In Latin America, Brazil harbors the most cases of human visceral leishmaniasis (HVL). Since the early 1980s, the disease has spread to the urban centers of the north, and now the south and west of Brazil; it reached São Paulo state in the southeast in 1996, and Presidente Prudente in the western region in 2010. Our aim was to describe the spatiotemporal analysis and environmental risk factors associated with the dispersion of VL in Presidente Prudente, an urban setting with recent transmission. Methods An entomological survey was carried out from 2009 to 2015. A canine visceral leishmaniasis (CVL) serosurvey was performed from 2010 to 2015 using enzyme-linked immunosorbent assays (ELISA), a dual-path platform CVL rapid test, and indirect fluorescent antibodies (IFAT). Data from HVL cases were obtained from the Municipal Surveillance Epidemiology Center from 2013 to 2017. Data on water drainage and forest fragments were obtained from public platforms and irregular solid-waste deposits were determined by monthly inspections of the urban area. Kernel density maps of the distribution of CVL were constructed. Results From 2009 to 2015, Lutzomyia longipalpis sand flies were found in all seven areas of Presidente Prudente. From 2010 to 2015, 40,309 dogs were serologically screened and 638 showed positive results, i.e. a prevalence rate of 1.6%. From 2013 to 2017, six human cases were diagnosed with a mortality rate of 33.3%. In 2015, 56 points of irregular solid-waste deposits were identified, predominantly in the neighborhoods. Three different hotspots of CVL showed an increased distribution of vectors, seropositive dogs, irregular solid-waste deposits, forest fragments and water drainage. Conclusions The use of tools that analyze the spatial distribution of vectors, canine and human VL as environmental risk factors were essential to identifying the areas most vulnerable to the spread or maintenance of VL. The results may help public health authorities in planning prevention and control measures to avoid expansion and future outbreaks.

Abstract Background In Brazil, members of the sand fly species complex Lutzomyia longipalpis transmit Leishmania infantum, a protist parasite that causes visceral leishmaniasis. Male Lu. longipalpis produce a sex pheromone that is attractive to both females and males. During a cluster randomised trial, to determine the combined effect of synthetic sex-aggregation pheromone and insecticide on Le. infantum transmission Lu. longipalpis had been continuously exposed to insecticide for 30 months. The objective of this study was to determine the effect of continuous exposure to the insecticides used in the trial on the susceptibility of Lu. longipalpis population. Methods During the trial the sand flies had been exposed to either lambda-cyhalothrin [pheromone + residual insecticide spray (PI)], deltamethrin [dog collars (DC)] or no insecticide [control (C)], for 30 months (November 2012 to April 2015). The insecticide treatment regime was kept in place for an additional 12 months (May 2015-April 2016) during this susceptibility study. Sand flies collected from the field were exposed to WHO insecticide-impregnated papers cyhalothrin (0.05%), deltamethrin (0.5%) and control (silicone oil) in a modified WHO insecticide exposure trial to determine their susceptibility. Results We collected 788 Lu. longipalpis using CDC-light traps in 31 municipalities across the three trial arms. Probit analysis showed that the knockdown times (KDTs) of Lu. longipalpis collected from the lambda-cyhalothrin exposed PI-arm [KDT50: 31.1 min, confidence interval (CI): 29.6–32.6 and KDT90: 44.2 min, CI: 42.1–46.7] were longer than the KDTs from the non-insecticide-treated C-arm (KDT50: 26.3 min, CI: 25.1–27.6 and KDT90: 38.2, CI: 36.5–40.2) (no-overlapping 95% CIs). KDTs of Lu. longipalpis collected from the deltamethrin exposed DC-arm had similar values (KDT50: 13.7 min, CI: 10.1–16.2 and KDT90: 26.7 min, CI: 21.8–30.6) to those for the C-arm (KDT50: 13.5 min; CI: 12.2–14.8 and KDT90: 23.2 min, CI: 21.4–25.4) (overlapping CIs). The wild-caught unexposed Lu. longipalpis (C-arm), took approximately twice as long to knock down as laboratory-colonised specimens for both insecticides. Conclusions Our study reveals slight changes in KDT, in sand flies after prolonged exposure to lambda-cyhalothrin in the presence of pheromone. These changes are not considered to have reached the reference levels indicative of resistance in sand flies suggesting that pheromone and insecticide treatment at the level indicated in this study do not constitute a significant risk of increased insecticide resistance. Prolonged exposure to deltamethrin in dog collars did not result in changes to KDT.

Abstract Background The leishmaniases are important neglected diseases caused by Leishmania spp. which are transmitted by sand flies, Lutzomyia longipalpis being the main vector of visceral leishmaniasis in the Americas. The methodologies for leishmaniasis control are not efficient, causing 1.5 million reported cases annually worldwide, therefore showing the need for development of novel strategies and interventions to control transmission of the disease. The bacterium Wolbachia pipientis is being used to control viruses transmitted by mosquitoes, such as dengue and Zika, and its introduction in disease vectors has been effective against parasites such as Plasmodium. Here we show the first successful establishment of Wolbachia into two different embryonic cell lines from L. longipalpis, LL-5 and Lulo, and analysed its effects on the sand fly innate immune system, followed by in vitro Leishmania infantum interaction. Results Our results show that LL-5 cells respond to wMel and wMelPop-CLA strains within the first 72 h post-infection, through the expression of antimicrobial peptides and inducible nitric oxide synthase resulting in a decrease of Wolbachia detection in the early stages of infection. In subsequent passages, the wMel strain was not able to infect any of the sand fly cell lines while the wMelPop-CLA strain was able to stably infect Lulo cells and LL-5 at lower levels. In Wolbachia stably infected cells, the expression of immune-related genes involved with downregulation of the IMD, Toll and Jak-Stat innate immune pathways was significantly decreased, in comparison with the uninfected control, suggesting immune activation upon Wolbachia transinfection. Furthermore, Wolbachia transinfection did not promote a negative effect on parasite load in those cells. Conclusions Initial strong immune responses of LL5 cells might explain the inefficiency of stable infections in these cells while we found that Lulo cells are more permissive to infection with Wolbachia causing an effect on the cell immune system, but not against in vitro L. infantum interaction. This establishes Lulo cells as a good system for the adaptation of Wolbachia in L. longipalpis.

Abstract Background The sand fly Lutzomyia longipalpis is the main vector of American visceral leishmaniasis, a disease caused by parasites of the genus Leishmania. Adults of this insect feed on blood (females only) or sugar from plant sources, but their digestion of carbohydrates is poorly studied. Beta-glycosides as esculin and amygdalin are plant compounds and release toxic compounds as esculetin and mandelonitrile when hydrolyzed. Beta-glucosidase and trehalase are essential enzymes in sand fly metabolism and participate in sugar digestion. It is therefore possible that the toxic portions of these glycosides, released during digestion, affect sand fly physiology and the development of Leishmania. Results We tested the oral administration to sand flies of amygdalin, esculin, mandelonitrile, and esculetin in the sugar meal. These compounds significantly decreased the longevity of Lutzomyia longipalpis females and males. Lutzomyia longipalpis adults have significant hydrolytic activities against esculin and feeding on this compound cause changes in trehalase and β-glucosidase activities. Female trehalase activity is inhibited in vitro by esculin. Esculin is naturally fluorescent, so its ingestion may be detected and quantified in whole insects or tissue samples stored in methanol. Mandelonitrile neither affected the amount of sugar ingested by sand flies nor showed repellent activity. Our results show that mandelonitrile significantly reduces the viability of L. amazonensis, L. braziliensis, L. infantum and L. mexicana, in a concentration-dependent manner. Esculetin caused a similar effect, reducing the number of L. infantum and L. mexicana. Female L. longipalpis fed on mandelonitrile had a reduction in the number of parasites and prevalence of infection after seven days of infection with L. mexicana, either by counting in a Neubauer chamber or by qPCR assays. Conclusions Glycosides have significant effects on L. longipalpis longevity and metabolism and also affect the development of parasites in culture and inside the insect. These observations might help to conceptualize new vector control strategies using transmission blocking sugar baits.

Sugar-rich food sources are essential for sandflies to meet their energy demands, achieving more prolonged survival. The digestion of carbohydrates from food is mainly realized by glycoside hydrolases (GH). To identify genes coding for α-glycosidases and α-amylases belonging to Glycoside Hydrolase Family 13 (GH13) and Glycoside Hydrolase Family 31 (GH31) in Lutzomyia longipalpis, we performed an HMMER search against its genome using known sequences from other dipteran species. The sequences retrieved were classified based on BLASTP best hit, analysis of conserved regions by alignment with sequences of proteins with known structure, and phylogenetic analysis comparing with orthologous proteins from other dipteran species. Using RT-PCR analysis, we evaluated the expression of GH13 and GH31 genes, in the gut and rest of the body of females, in four different conditions: non-fed, sugar-fed, blood-fed, and Leishmania mexicana infected females. L. longipalpis has GH13/31 genes that code for enzymes involved in various aspects of sugar metabolism, as carbohydrate digestion, storage, and mobilization of glycogen reserves, proteins involved in transport, control of N-glycosylation quality, as well as others with a putative function in the regulation of myogenesis. These proteins are representatives of GH13 and GH31 families, and their roles seem to be conserved. Most of the enzymes seem to be active with conserved consense sequences, including the expected catalytic residues. α-amylases also demonstrated the presence of calcium and chloride binding sites. L. longipalpis genome shows an expansion in the α-amylase gene family, with two clusters. In contrast, a retraction in the number of α-glucosidases occurred. The expansion of α-amylases is probably related to the specialization of these proteins for different substrates or inhibitors, which might correlate with the higher diversity of plant foods available in the natural habitat of L. longipalpis. The expression of α-glucosidase genes is higher in blood-fed females, suggesting their role in blood digestion. Besides that, in blood-fed females infected with the parasite Leishmania mexicana, these genes were also modulated. Glycoside Hydrolases from families 13 and 31 are essential for the metabolism of L. longipalpis, and GH13 enzymes seem to be involved in the interaction between sandflies and Leishmania.

Abstract Lutzomyia longipalpis is the most important vector of Leishmania infantum, the etiological agent of visceral leishmaniasis (VL) in the New World. It is a permissive vector susceptible to infection with several Leishmania species. One of the advantages that favors the study of this sand fly is the possibility of colonization in the laboratory. For this reason, several researchers around the world use this species as a model for different subjects including biology, insecticides testing, host-parasite interaction, physiology, genetics, proteomics, molecular biology, and saliva among others. In 2003, we published our first review (Soares & Turco 2003) on this vector covering several aspects of Lu. longipalpis. This current review summarizes what has been published between 2003-2020. During this period, modern approaches were incorporated following the development of more advanced and sensitive techniques to assess this sand fly.

Sugar-rich food sources are essential for sandflies to meet their energy demands, achieving more prolonged survival. The digestion of carbohydrates from food is mainly realized by glycoside hydrolases (GH). To identify genes coding for α-glycosidases and α-amylases belonging to Glycoside Hydrolase Family 13 (GH13) and Glycoside Hydrolase Family 31 (GH31) in Lutzomyia longipalpis , we performed an HMMER search against its genome using known sequences from other dipteran species. The sequences retrieved were classified based on BLASTP best hit, analysis of conserved regions by alignment with sequences of proteins with known structure, and phylogenetic analysis comparing with orthologous proteins from other dipteran species. Using RT-PCR analysis, we evaluated the expression of GH13 and GH31 genes, in the gut and rest of the body of females, in four different conditions: non-fed, sugar-fed, blood-fed, and Leishmania mexicana infected females. L. longipalpis has GH13/31 genes that code for enzymes involved in various aspects of sugar metabolism, as carbohydrate digestion, storage, and mobilization of glycogen reserves, proteins involved in transport, control of N-glycosylation quality, as well as others with a putative function in the regulation of myogenesis. These proteins are representatives of GH13 and GH31 families, and their roles seem to be conserved. Most of the enzymes seem to be active with conserved consense sequences, including the expected catalytic residues. α-amylases also demonstrated the presence of calcium and chloride binding sites. L. longipalpis genome shows an expansion in the α-amylase gene family, with two clusters. In contrast, a retraction in the number of α-glucosidases occurred. The expansion of α-amylases is probably related to the specialization of these proteins for different substrates or inhibitors, which might correlate with the higher diversity of plant foods available in the natural habitat of L. longipalpis. The expression of α-glucosidase genes is higher in blood-fed females, suggesting their role in blood digestion. Besides that, in blood-fed females infected with the parasite Leishmania mexicana , these genes were also modulated. Glycoside Hydrolases from families 13 and 31 are essential for the metabolism of L. longipalpis , and GH13 enzymes seem to be involved in the interaction between sandflies and Leishmania. [ABSTRACT FROM AUTHOR]

The sand fly Lutzomyia longipalpis is the main vector of Leishmania infantum in Brazil. Synthetic male-produced sex/aggregation pheromone co-located with micro-encapsulated λ-cyhalothrin in chicken sheds can significantly reduce canine infection and sand fly densities in a lure-and-kill strategy. In this study, we determined if insecticide-impregnated netting (IN) could replace insecticide residual spraying (IRS). We compared numbers of Lu. longipalpis attracted and killed in experimental and real chicken sheds baited with pheromone and treated with a 1 m2 area of either insecticide spray or netting. First, we compared both treatments in experimental sheds to control mortality established from light trap captures. We then compared the long-term killing effect of insecticide spray and netting, without renewal, in experimental sheds over a period of 16 weeks. Finally, a longitudinal intervention study in real chicken sheds compared the numbers and proportions of Lu. longipalpis collected and killed before and after application of both treatments. In Experiment 1, a higher proportion of males and females captured in IRS- and IN-treated sheds were dead at 24 h compared to controls (P

Background: Leishmania infantum is the etiological agent of visceral leishmaniasis (VL) in the New World, where the sand fly Lutzomyia longipalpis and domestic dogs are considered the main vector and host reservoirs, respectively. Systemic insecticides have been studied as an alternative to control vector-borne diseases, including VL. Fluralaner, an isoxazoline class compound, is a systemic insecticide used in dogs, with proven efficiency against different species of phlebotomine sand flies. However, to date no studies have demonstrated the efficacy of fluralaner on Lu. longipalpis. The aim of this study was to evaluate the insecticidal effect of fluralaner (Bravecto®) on the sand fly Lu. longipalpis after blood meal in treated dogs. Methods: Healthy mongrel dogs (n = 8) were recruited from the Zoonoses Control Center in the city of Natal, Rio Grande do Norte, Brazil, and randomized into two groups: fluralaner treated (n = 4) and non-treated control (n = 4). Colony-reared female specimens of Lu. longipalpis (n = 20) were allowed to feed on all dogs for 40 min before treatment (for fluralaner-treated dogs), at day 1 after treatment and then monthly until 1 year post-treatment. Results: In the treatment group, there was 100% mortality of Lu. longipalpis for up to 5 months after treatment initiation, decreasing to 72.5% at 6 months post-treatment initiation. The efficacy of fluralaner ranged from 100% at day 1 (P = 0.0002) to 68% (P = 0.0015) at 6 months, decreasing to 1.4% at 1 year post-treatment. Sand fly mortality carried out blood meal in non-treated control dogs remained constant at ≤ 15%. Conclusions: Taken together, our results suggest that fluralaner may be used as a control strategy for VL in dogs in VL endemic areas. [ABSTRACT FROM AUTHOR]

Abstract INTRODUCTION In Bolivia, before 1982 there were no records of visceral leishmaniasis (VL) cases that would allow us to review and describe the temporospatial occurrence of VL by ecoregions in provinces and departments of Bolivia to evaluate its impact on public health, risk of outbreaks, or dispersion. METHODS This update on VL in Bolivia is based on research, reviews, and retrospective literature analyses of online data and libraries and institutional reports, from 1939 to the present. RESULTS In Bolivia, 56 cases of VL have been reported. Until 2014, only three endemic departments had been identified (La Paz, Santa Cruz, and Tarija). Since then, further cases have been recorded in Pando, Cochabamba, and Beni, and in Chuquisaca in 2015. In Yungas, a VL focus was confirmed by isolating and comparing parasites from human and dog cases, and from the Lu. longipalpis vector. VL cases from seven departments, involving 12 different ecoregions were located within the Amazon and Plata basins. CONCLUSIONS We confirmed that dogs are its primary reservoir, and Lutzomyia longipalpis is its main vector (currently dispersed in six departments). The primary vectors in areas where Lutzomyia longipalpis is absent are Migonemyia migonei and Lutzomyia cruzi.

Abstract INTRODUCTION: Leishmaniasis constitutes a serious but neglected tropical disease. Recently, socio-environmental, biological and physical changes have altered the range of leishmaniasis, causing it to spread into urban areas. In Minas Gerais, the disease is endemic, exhibiting regional differences and reaching urban centers. The purpose of this study was to evaluate entomological aspects related to the ecoepidemiology of leishmaniasis in Itaúna. METHODS: Monthly catches with HP traps were carried out from June 2017 to May 2018, in three ecological areas (urban, rural, and forest). The adaptability of the species to anthropic environments was assessed using the synanthropy index (SI). RESULTS: We collected 1306 specimens of phlebotomine sand flies. Of the species of medical importance, Lutzomyia longipalpis, the vector of Leishmania infantum, represented 90.4% of the specimens identified at species level (n=1260). Nyssomyia whitmani, the vector of Leishmania braziliensis, represented 1.6% of the total. Lu. longipalpis displayed an SI of +95.8, a value that denotes a marked preference for human environments. For Ny. whitmani, the SI was -25, expressing the tendency of this species to occupy uninhabited areas. The population of the three most numerous species captured increased with rain, high temperatures, and high relative humidity. Although captured at low numbers, Ny. whitmani showed a different profile concerning the climate variables analyzed. CONCLUSIONS: Understanding the epidemiology of the disease may assist the health services in formulating effective control measures for improving community health and contributing to the establishment of a dynamic relationship and a global awareness of the health/disease process.

Dogs are considered the main reservoirs of visceral leishmaniasis for humans, which also present a chronic and severe clinical picture when affected. The objective of the present report was to describe a canine visceral leishmaniasis case diagnosed in Londrina, an indene city, and its investigation. A street animal with extensive dermatological lesions, onychogryphosis, mild anemia and leukopenia was attended at a veterinary hospital in Londrina, where positivity was reported for Leishmania spp. in serological tests. Cytology was positive in bone marrow, PCR and parasite culture were positive in skin, spleen, liver, lymph node and bone marrow, and DNA sequencing confirmed the species of the parasite as L. (L.) infantum. The official diagnosis was made by the Central Laboratory of Paraná (LACEN), and through an official report, an investigation of the case was started for the confirmation of autochthony. An active search for the vector and other canine cases in the neighborhood was carried out along with a search for information on the origin of the animal in question. However, the species, Lutzomyia longipalpis, new canine cases, or origin of the sick animal were not identified. Although, the present case cannot be confirmed as autochthonous, we suggest that it is necessary to disseminate the present report to serve as a warning to veterinarians and other public health professionals in the northern region of Paraná to be attentive to suspicious cases and to not fail to investigate these cases to the end.

Abstract Background Lutzomyia longipalpis is the main vector of Leishmania infantum, the agent of canine and human visceral leishmaniosis in the Americas. Considering that the dog is the main domestic host of the parasite, repellent treatment is a measure that might contribute to the prevention of canine visceral leishmaniosis. The repellent efficacy of a single treatment of a new spot-on topical combination of fipronil and permethrin (Frontline Tri-Act®, Merial, now part of Boehringer-Ingelheim) to repel Lu. longipalpis sand flies was evaluated. Methods Sixteen healthy Beagle dogs, eight females and eight males, weighing 8.4–14.4 kg, and 2 to 4 years-old were included in the study. Animals were blocked on decreasing body weight and randomly allocated within the blocks to one of two treatment groups of eight animals each. Dogs in Group 1 were untreated and Dogs in Group 2 were treated with a combination containing 67.6 mg/ml fipronil + 504.8 mg/ml permethrin (Frontline Tri-Act®) once on Day 0. Sand fly exposures were performed on Days 1, 14, 21 and 30 with Lu. longipalpis female sand flies. After 65 (± 15 min), sand flies were assessed for engorgement status. Results The percent repellency of the treated group compared to the untreated control group was 95.7, 94.3, 81.7 and 72.2% for exposure days 1, 14, 21 and 30, respectively. The two treatment groups were significantly different for all exposure days (P ≤ 0.016 for days 1, 14, 21 and 30). No adverse reactions were observed during the study. Conclusion A single topical administration of a new combination of fipronil and permethrin demonstrated a significant repellent effect against Lu. longipalpis bites as soon as it was applied on the dogs and its repellent efficacy lasted for 4 weeks with results greater than 80% for 3 weeks. The results suggest that in endemic areas the regular application of the new combination could contribute to protect dogs from Leishmania infection and therefore serve as an additional tool for the prevention of canine visceral leishmaniosis.

Background: In South America, cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) are emerging diseases, expanding in the border area of Argentina, Brazil and Paraguay. Outbreaks of CL were reported since the 1990s, with Nyssomyia whitmani as the main vector in this region. Regarding VL, urban reports started in 2010 with Lutzomyia longipalpis as the main vector. The aim of this study was to evaluate environmental determinants related to the main vectors of leishmaniasis, to contribute to the prevention and control response to the emergence of VL and CL in the Argentina-Brazil-Paraguay border region. Methods: The cross-sectional survey includes two cities and two close rural areas in the Argentinean Northeast Region, between November 2014 and January 2015, with a total of 95 sampling sites. REDILA-BL traps were set for three consecutive nights, and a total of 68 meso- and microscale environmental and landscape characteristics were surveyed. The association between vector abundance with different variables was evaluated using a generalized linear model with zero-inflated negative binomial distribution. We analyzed females for detection of Leishmania DNA. Results: The analysis for Lu. longipalpis indicates an excess of absences when the mean NDWI around the sites were higher. The abundance of Lu. longipalpis at mesoscale level was higher when more urban services were present, and when blood sources such as chickens or dogs at the microscale level were present. For Ny. whitmani, no variable was found to be associated with the absences, while its abundance increased in association with the following variables: percentage of tree cover, presence of garbage collection service, hosted people and, at microscale, the presence of poultry. Leshmania infantum DNA was detected in 2/49 (4%) Lu. longipalpis. Conclusions: The abundance of both species is influenced by variables at different scales, their influence probably has a hierarchy and they are acting on different aspects of the biology of these vectors. The urban spatial segregation of Lu. longipalpis and the peri-urban and rural segregation of N. whitmani increase the risk of VL and CL. The selection of the better variables for each scale will allow the design of appropriate control strategies depending on species. [ABSTRACT FROM AUTHOR]

An epidemiological Leishmania spp. and entomological Phlebotomine sandflies survey was performed in cat shelters at leishmaniasis endemic area of Brazil. Blood and conjunctival swab (CS) samples were collected from 94 cats in two animal protection shelters. These samples were subjected to serological tests using the indirect immunofluorescence antibody test (IFAT) and indirect enzyme‐linked immunosorbent assay (ELISA) and to molecular test by polymerase chain reaction (PCR). In addition, a Phlebotomine sandflies survey was performed in the same shelters. The analyses revealed a positivity of 31.91% (30/94) through ELISA and 29.79% (28/94) through IFAT. The two serological tests showed a positive association with perfect agreement (k = 0.925). None of the cats were positive by Leishmania spp. DNA. One Lutzomyia (Lutzomyia) longipalpis male was found in one of the cat shelters. The results and the implications of our findings are discussed below. [ABSTRACT FROM AUTHOR]

This study evaluated the implications of clinically acquired miltefosine resistance (MIL-R) by assessing virulence in mice and sand flies to reveal the potential of MIL-R strains to circulate. Experimental infections with the MIL-R clinical Leishmania infantum isolate MHOM/FR/2005/LEM5159, having a defect in the LiROS3 subunit of the MIL-transporter, and its syngeneic experimentally reconstituted MIL-S counterpart (LEM5159 LiROS3 ) were performed in BALB/c mice and Lutzomyia longipalpis and Phlebotomus perniciosus sand flies. In mice, the amastigote burdens in liver and spleen were compared microscopically using Giemsa smears and by bioluminescent imaging. During the sand fly infections, the percentage of infected flies, parasite load, colonization of the stomodeal valve and metacyclogenesis were evaluated. The stability of the MIL-R phenotype after sand fly and mouse passage was determined as well. The fitness of the MIL-R strain differed between the mouse and sand fly infection model. In mice, a clear fitness loss was observed compared to the LiROS3 -reconstituted susceptible strain. This defect could be rescued by episomal reconstitution with a wildtype LiROS3 copy. However, this fitness loss was not apparent in the sand fly vector, resulting in metacyclogenesis and efficient colonization of the stomodeal valve. Resistance was stable after passage in both sand fly and mouse. The natural MIL-R strain is significantly hampered in its ability to multiply and cause a typical visceral infection pattern in BALB/c mice. However, this LiROS3-deficient strain efficiently produced mature infections and metacyclic promastigotes in the sand fly vector highlighting the transmission potential of this particular MIL-R clinical Leishmania strain. Image 1 • A clinical MIL-R L. infantum strain displays a loss-of-fitness in the mammalian host. • ROS3-deficiency is compatible with efficient transmission by two sand fly species. • Resistance is stable after mouse and sand fly passage. • Transmission of this clinical MIL-R strain is a risk for immunocompromised patients. [ABSTRACT FROM AUTHOR]

Background: Since the introduction of miltefosine (MIL) as first-line therapy in the kala-azar elimination programme in the Indian subcontinent, treatment failure rates have been increasing. Since parasite infectivity and virulence may become altered upon treatment relapse, this laboratory study assessed the phenotypic effects of repeated in vitro and in vivo MIL exposure. Methods: Syngeneic Leishmania donovani lines either or not exposed to MIL were compared for drug susceptibility, rate of promastigote multiplication and metacyclogenesis, macrophage infectivity and behaviour in the sand fly vector, Lutzomyia longipalpis. Results: Promastigotes of both in vitro and in vivo MIL-selected strains displayed a slightly reduced drug susceptibility that was associated with a reduced MIL-accumulation linked to a lower copy number (disomic state) of chromosome 13 harboring the miltefosine transporter (LdMT) gene. In vitro selected promastigotes showed a lower rate of metacyclogenesis whereas the in vivo derived promastigotes displayed a moderately increased growth rate. Repeated MIL exposure did neither influence the parasite load nor metacyclogenesis in the sand fly vector. Conclusions: Recurrent in vitro and in vivo MIL exposure evokes a number of very subtle phenotypic and genotypic changes which could make promastigotes less susceptible to MIL without attaining full resistance. These changes did not significantly impact on infection in the sand fly vector. [ABSTRACT FROM AUTHOR]

Abstract Background The leishmaniases are a group of diseases caused by protozoans of the genus Leishmania, which are transmitted by the bite of phlebotomine sand flies. In the New World, Lutzomyia longipalpis is the most important vector of visceral leishmaniasis and is a proven vector for Leishmania infantum chagasi in Brazil. During development within the vector, Leishmania can interact with a variety of microorganisms such as fungi and bacteria. The presence of bacteria in the midgut of sand flies can influence the development and survival of the parasite. Results The bacteria-targeted metagenomic analysis revealed different community compositions between the distinct physiological stages of those tested. The amplicon-oriented metagenomic profiling revealed 64 bacterial genera and 46 families. By crossing the taxa indices from each experimental condition a core composed of 6 genera was identified (Enterobacter, Serratia, Stenotrophomonas, Enhydrobacter, Pseudomonas and Chryseobacterium). Conclusions The observed dynamic nature of the bacterial community expands the knowledge pertaining to the tripartite host-microbiota-pathogen interactions. Further studies addressing how laboratory and field collected communities differ are critical to successfully develop control strategies based on bacterial symbionts and paratransgenesis, as already tested in other arthropod vectors.

Abstract Background Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes. Its species-specific polymorphisms are found mainly in the sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Leishmania amazonensis is one of the most important species causing human cutaneous leishmaniasis in the New World. Here, we describe LPG intraspecific polymorphisms in two Le. amazonensis reference strains and their role during the development in three sand fly species. Results Strains isolated from Lutzomyia flaviscutellata (PH8) and from a human patient (Josefa) displayed structural polymorphism in the LPG repeat units, possessing side chains with 1 and 2 β-glucose or 1 to 3 β-galactose, respectively. Both strains successfully infected permissive vectors Lutzomyia longipalpis and Lutzomyia migonei and could colonize their stomodeal valve and differentiate into metacyclic forms. Despite bearing terminal galactose residues on LPG, Josefa could not sustain infection in the restrictive vector Phlebotomus papatasi. Conclusions LPG polymorphisms did not affect the ability of Le. amazonensis to develop late-stage infections in permissive vectors. However, the non-establishment of infection in Ph. papatasi by Josefa strain suggested other LPG-independent factors in this restrictive vector.